Antibodies to ticagrelor and methods of use

ABSTRACT

The disclosure generally provides antibodies and antigen binding fragments of antibodies that bind ticagrelor and metabolites of ticagrelor. The disclosure also provides compositions comprising the antibodies, nucleic acid molecules encoding the antibodies, methods of treating a patient comprising administering the antibodies, and methods of making and using the antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/035,954, filed Jul. 16, 2018; now U.S. Pat. No. 10,954,308, issuedMar. 23, 2021, which is a divisional of U.S. application Ser. No.15/966,313, filed Apr. 30, 2018; now abandoned, which is a continuationof U.S. application Ser. No. 14/871,111, filed Sep. 30, 2015, now U.S.Pat. No. 9,982,061, issued May 29, 2018; which claims the benefit ofpriority to U.S. Provisional Application No. 62/114,931, filed Feb. 11,2015; and U.S. Provisional Application No. 62/058,458, filed Oct. 1,2014, the contents of each of which are incorporated by reference intheir entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named PHAS-035_06US_SeqList_ST25.txt,which was recorded on Feb. 16, 2021 and is 34 kB in size, are herebyincorporated by reference in their entireties.

BACKGROUND

Ticagrelor (BRILINTA™, BRILIQUE™) is an orally activecyclopentyltriazolopyrimidine, a selective and reversibly bindingadenosine diphosphate (ADP) receptor antagonist. In patients with acutecoronary syndromes (ACS), ticagrelor 90 mg twice daily in combinationwith low-dose aspirin is approved to reduce major cardiovascular (CV)events. Ticagrelor acts via a dual pathway which mediates bothantiplatelet effects (P2Y₁₂) and an enhanced adenosine response (ENT-1)(Cattaneo M et al 2014. J Am Coll Cardiol. 63(23):2503-9). While not yetapproved indications, ongoing and planned studies are evaluatingticagrelor for reduction of major CV events in patients with priormyocardial infarction, established peripheral arterial disease, andacute stroke, as well as patients with diabetes and confirmed coronaryatherosclerosis.

Ticagrelor has two primary metabolites, ticagrelor active metabolite(TAM) and ticagrelor inactive metabolite (TIM) (Teng et al 2010 DrugMetab. and Dispos. 38:1514-1521). TAM, also known as AR-C124910XX, isthe main circulating metabolite of ticagrelor and is equally effectivein P2Y₁₂ antagonist activity. TAM typically present at about 30-40% ofthe parent ticagrelor concentration in patients on BRILINTA/BRILIQUE.Ticagrelor and TAM have circulating half-lives of 8 and 12 hoursrespectively. TIM, also known as AR-C133913XX, is inactive againstP2Y₁₂, constitutes <10% of the parent ticagrelor, is undetectable after8 hours, and is the main metabolite excreted via the urine.

The PLATelet inhibition and patient Outcomes (PLATO) trial hasdemonstrated greater efficacy of ticagrelor without an increase in totalmajor bleeding when compared with clopidogrel in a broad ACS patientpopulation (UA, NSTEMI, STEMI) regardless of management strategy(medically or invasively managed) (Wallentin et al 2009 NEJM 361(11):1045-1057). As with all antiplatelet agents, however, there existsthe potential for bleeding in patients using ticagrelor. There arelimited treatment options if severe bleeding occurs in patients on dualantiplatelet therapy (DAPT). If a bleeding event occurs in a patient onDAPT, platelet transfusions or administration of coagulation factors maybe use in an attempt to augment haemostasis. However, currently noclinical data exist that evaluates the haemostatic benefit of platelettransfusions or use of recombinant Factor VIIa after or during a majorbleeding event in subjects on ticagrelor (Daldn M et al 2013 JCardiothorac Vase Anesth. 27(5):e55-7).

Accordingly, the availability of an antidote, such as aticagrelor-specific neutralizing antibody, would allow better clinicalmanagement of the balance between the desired antithrombotic effectversus control of bleeding. Since ticagrelor is the only marketedreversibly binding platelet inhibitor an antibody may provide reversalof platelet inhibition without the need for fresh platelet transfusions,thus avoiding hazards associated with platelet transfusions. Theavailability of an agent that overcomes the inhibition of ADP-inducedplatelet aggregation associated with ticagrelor and TAM would fulfill animportant unmet clinical need, for example in patients who experiencemajor bleeding or who require urgent surgery.

SUMMARY OF THE DISCLOSURE

In an aspect the disclosure relates to an antibody that specificallybinds a cyclopentyltriazolopyrimidine compound of the Formula (Ia):

-   -   wherein    -   R₁ is selected from the group consisting of C₁-C₆ alkoxy and        C₁-C₆ alkylthio;    -   R₂ is selected from the group consisting of H, C₁-C₆ alkyl,        substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and substituted C₃-C₆        cycloalkyl; and    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl,        C₁-C₆ alkoxy, and C₁-C₆ alkanol.

In some embodiments, the antibody binds to an epitope within the portionof the compounds identified by brackets as Formula (IIa)

-   -   wherein R₁, R₂, and R₃ are defined as above.

In further embodiments, the antibody binds to an epitope within theportion of the compounds identified by brackets as Formula (IIIa)

-   -   wherein R₂, and R₃ are defined as above and R′₁ is selected from        the group consisting of C₁-C₄ alkyl.

In further embodiments, the antibody binds to a compound selected fromthe group consisting of

In further embodiments of the above aspects and embodiments, theantibody or a fragment thereof comprises a heavy chain variable region(VH) sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO: 12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ IDNO:62, and SEQ ID NO:72; and a light chain variable region (VL) sequenceselected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ IDNO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, and SEQID NO:77. In some embodiments, the antibody comprises a combination ofVH and VL sequences selected from the group consisting of SEQ ID NO:2and SEQ ID NO:7; SEQ ID NO: 12 and SEQ ID NO: 17; SEQ ID NO:22 and SEQID NO:27; SEQ ID NO:32 and SEQ ID NO:37; SEQ ID NO:42 and SEQ ID NO:47;SEQ ID NO:52 and SEQ ID NO:57; SEQ ID NO:62 and SEQ ID NO: 67; and SEQID NO:72 and SEQ ID NO:77. In further embodiments, the antibodycomprises a combination of VH and VL selected from the group consistingof SEQ ID NO:52 and SEQ ID NO:57; SEQ ID NO:62 and SEQ ID NO:67; and SEQID NO:72 and SEQ ID NO:77.

In further embodiments of the above aspects and embodiments, theantibody or a fragment thereof comprises framework regions (FR) andcomplementarity-determining regions (CDRs) 1, 2, and 3 of a heavy chainvariable region and a light chain variable region, wherein the CDR1,CDR2, and CDR3 sequences of the heavy chain variable region comprise,SEQ ID NO:3 (CDR1), SEQ ID NO:4 (CDR2), and SEQ ID NO:5 (CDR3); SEQ IDNO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3); SEQ IDNO:23 (CDR1), SEQ ID NO:24 (CDR2), and SEQ ID NO:25 (CDR3); SEQ ID NO:33(CDR1), SEQ ID NO:34 (CDR2), and SEQ ID NO:35 (CDR3); SEQ ID NO:43(CDR1), SEQ ID NO:44 (CDR2), and SEQ ID NO:45 (CDR3); SEQ ID NO:53(CDR1), SEQ ID NO:54 (CDR2), and SEQ ID NO:55 (CDR3); SEQ ID NO:63(CDR1), SEQ ID NO:64 (CDR2), and SEQ ID NO:65 (CDR3); or SEQ ID NO:73(CDR1), SEQ ID NO:74 (CDR2), and SEQ ID NO:75 (CDR3); and wherein theCDR1, CDR2, and CDR3 sequences of the light chain variable regioncomprise, SEQ ID NO:8 (CDR1), SEQ ID NO:9 (CDR2), and SEQ ID NO: 10(CDR3); SEQ ID NO: 18 (CDR1), SEQ ID NO: 19 (CDR2), and SEQ ID NO:20(CDR3); SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2), and SEQ ID NO:30(CDR3); SEQ ID NO:38 (CDR1), SEQ ID NO:39 (CDR2), and SEQ ID NO:40(CDR3); SEQ ID NO:48 (CDR1), SEQ ID NO:49 (CDR2), and SEQ ID NO:50(CDR3); SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60(CDR3); SEQ ID NO:68 (CDR1), SEQ ID NO:69 (CDR2), and SEQ ID NO:70(CDR3); or SEQ ID NO:78 (CDR1), SEQ ID NO:79 (CDR2), and SEQ ID NO:80(CDR3). In further embodiments, the antibody comprises a combination ofCDR regions selected from the group consisting of: SEQ ID NO:53 (VHCDR1), SEQ ID NO:54 (VH CDR2), SEQ ID NO:55 (VH CDR3), SEQ ID NO:58 (VLCDR1), SEQ ID NO:59 (VL CDR2), and SEQ ID NO:60 (VL CDR3); SEQ ID NO:63(VH CDR1), SEQ ID NO:64 (VH CDR2), SEQ ID NO:65 (VH CDR3), SEQ ID NO:68(VL CDR1), SEQ ID NO:69 (VL CDR2), and SEQ ID NO:70 (VL CDR3); and SEQID NO:73 (VH CDR1), SEQ ID NO:74 (VH CDR2), SEQ ID NO:75 (VH CDR3), SEQID NO:78 (VL CDR1), SEQ ID NO:79 (VL CDR2), and SEQ ID NO:80 (VL CDR3).

In the above aspect and embodiments, the antibody is selected from apolyclonal antibody, a monoclonal antibody, a humanized antibody, ahuman antibody, a single chain Fv (scFv), a single domain antibody, aFab, a F(ab′)₂, a single chain diabody, an antibody mimetic, and anantibody variable domain.

In some embodiments, the antibody comprises a scFv. In some embodiments,the antibody comprises a Fab.

In some embodiments, the antibody described above binds to ticagrelor orticagrelor active metabolite (TAM).

In further embodiments, the antibody binds ticagrelor or a metabolite orderivative thereof with an IC₅₀ of about 200 nM or lower. In yet furtherembodiments, the antibody binds ticagrelor or a metabolite or derivativethereof with an IC₅₀ of about 100 nM to about 1 nM, or with an IC₅₀ ofabout 10 nM to about 1 nM.

In further embodiments the antibody binds ticagrelor or a metabolite orderivative thereof with a K_(D) of about 50 nM or lower. In yet furtherembodiments, the antibody binds ticagrelor or a metabolite or derivativethereof with a K_(D) in a range of about 250 pM to about 1 pM, or in arange of about 100 pM to about 1 pM.

In further embodiments the antibody binds ticagrelor or a metabolite orderivative thereof and does not bind to a compound selected from thegroup consisting of fenofibrate, nilvadipine, cilostazol, bucladesine,regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid,simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate,adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosinetriphosphate. In embodiments, the antibody does not inhibit the activityof a compound selected from the group consisting of fenofibrate,nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide,cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole,adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeSadenosine diphosphate, and 2-MeS adenosine triphosphate. In furtherembodiments, the antibody exhibits an IC₅₀ of at least about 1000 μM fora compound selected from the group consisting of fenofibrate,nilvadipine, cilostazol, bucladesine, regadenoson, cyclothiazide,cyfluthrin, lovastatin, linezolid, simvastatin, cangrelor, pantoprazole,adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeSadenosine diphosphate, and 2-MeS adenosine triphosphate.

In some embodiments, the antibody has an in vivo half-life of about 4-12hours. In specific embodiments the antibody has an in vivo half-life ifabout 12 hours.

In some embodiments, the antibody neutralizes the antiplatelet effect ofticagrelor or the active metabolite of ticagrelor. In furtherembodiments, the antibody neutralizes the antiplatelet effect ofticagrelor or the active metabolite of ticagrelor within about 60minutes of administration.

In some embodiments, the antibody has an off-rate for ticagrelor or theactive metabolite of ticagrelor that allows for continuation ofcommencement of a therapy comprising ticagrelor.

In other aspects, the disclosure provides a method of treating acutebleeding in a patient who is in need of treatment, comprisingadministering to the patient an effective amount of the antibodydisclosed herein. In some embodiments of the method the patient hasundergone or is undergoing a surgical procedure and who has beenadministered ticagrelor. In some embodiments of the method the patientis in need of urgent care and/or emergency trauma management.

Other aspects of the disclosure provide for a composition comprising theantibody of any of the preceding aspects and embodiments in combinationwith a pharmaceutically acceptable carrier.

Some additional aspects provide a nucleic acid molecule comprising anucleotide sequence encoding an antibody according to of any of thepreceding claims. In some embodiments the nucleic acid moleculecomprises SEQ ID NO: 1, SEQ ID NO:6, SEQ ID NO: 11, SEQ ID NO:16, SEQ IDNO:21, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41, SEQ IDNO:46, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:66, SEQ IDNO:71, and SEQ ID NO: 76.

Further aspects of the disclosure provide for compositions, vectors, andhost cells that may comprise at least one nucleic acid moleculedisclosed herein. In some embodiments the compositions, vectors, andhost cells comprise a first nucleic acid molecule and a second nucleicacid molecule that encode one or more of the proteins disclosed herein.

Other aspects will be apparent to one of skill in the art upon review ofthe description and exemplary depictions that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the disclosure, there are depicted inthe drawings certain aspects of the disclosure. However, the disclosureis not limited to the precise arrangements and instrumentalities of theaspects depicted in the drawings.

FIG. 1 depicts PK/PD modelling of ticagrelor-neutralizing Fab based onPhase III PLATO data. Following a ticagrelor 180-mg loading dose and 90mg twice daily, neutralizing Fab is added at time zero. Patients arerestarted on ticagrelor on day 1. The Fab is predicted to rapidlyneutralize ticagrelor and the TAM, thus restoring platelet aggregationin 99% of patients. The ‘hill’ between days 0 and 1 represents theredistribution of ticagrelor from other tissues in the 1% of patients inwhom ticagrelor is cleared more slowly.

FIG. 2A-C—haptans and Fab specificity. (A) Provides the chemicalstructure of ticagrelor, ticagrelor active metabolite (TAM), ticagrelorinactive metabolite (TIM) and adenosine. The unique R groups,di-fluorophenyl-cyclopropyl and thiopropyl substituents, are highlightedwith a dotted line. (B) Specificity profile for TICA0072. (C)Specificity profile for TICA0212 (MEDI 2452). Specificity profilesinclude ticagrelor, TAM, TIM, adenosine, ADP, ATP and three of thetwelve related compounds of FIG. 4 (for clarity; no binding was detectedto any of the twelve compounds at concentrations up to 0.1 mM). Data ismean and SEM for three triplicates.

FIG. 3 illustrates correlation of scFv binding to biotinylated linkerticagrelor (x-axis) and binding to biotinylated linker ticagrelor in a50-fold excess of unmodified ticagrelor (y-axis). The inhibition of scFvbinding in the presence of excess unmodified ticagrelor is shown withlines for 0%, 50%, 80% and 90% inhibition.

FIG. 4 shows compounds identified with some degree of 2D, 3D orelectrostatic similarity to ticagrelor

FIG. 5A-F provides Selectivity studies for TICA0049 and TICA0072 Fab.a-c Competition of TICA0049 Fab binding to biotinylated ticagrelor bycompounds listed. d-f Competition of TICA0072 Fab binding tobiotinylated ticagrelor by compounds listed. Data DMSO normalised.

FIG. 6 provides competition curves for parent TICA0072 and optimizedvariants TICA0152, TICA0162 and TICA0212 Fabs in the second generationepitope competition assay.

FIG. 7A-F shows the results of Selectivity studies for TICA0162 andTICA0212 Fab. a-c Competition of TICA0162 Fab binding to biotinylatedticagrelor by compounds listed. d-f Competition of TICA0212 Fab bindingto biotinylated ticagrelor by compounds listed. Data DMSO normalised.

FIG. 8A-C shows the results of TICA0212/MEDI2452 or TICA0072concentration dependent reversal of ticagrelor. (A) TICA0212/MEDI2452 1uM ticagrelor (A) or 1 uM TAM (e) mediated inhibition of 20 μMADP-induced aggregation. (B) TICA0212/MEDI2452 shows reduction of freeticagrelor concentration in plasma in the presence of 1 uM ticagrelor.Mean (n=5)±standard error of the mean. (C) TICA0072 reversal ofticagrelor and TAM inhibition of P2Y₁₂ signalling.

FIG. 9 shows the results of TICA0212, 250 mg/kg, mediated reversal ofADP-induced whole blood aggregation ex vivo after dosing to ticagrelortreated mice.

FIG. 10A-B shows partial views of the crystal structures of TICA0072 (A)and TICA0212/MED12452 (B) in complex with ticagrelor. The Fabs are shownin ribbon representation with amino acid residues within 7 Å fromticagrelor shown as sticks. Some main chain atoms were omitted forclarity. Light chains are shown in beige and heavy chain in light blue.CDR3s from both chains are coloured green. VH CDR3 could not be modelledin the TICA0072 structure and a tentative location is drawn as a dashedline. The orange arrow indicates the shift in V_(L) CDR3 observed inTICA0212/MEDI2452 compared to TICA0072. Residues are number followingkabat and are prefixed with L or H to indicate light or heavy chain.

FIG. 11A-B shows reversal of ADP-induced whole blood aggregation exvivo. (A) Individual data for each treatment group post stop ofticagrelor infusion. Vehicle control (▪), ticagrelor alone (●),ticagrelor+TICA0212/MEDI2452 (□) and ticagrelor+isotype control (A). Barrepresents mean data (n=4). AU=aggregation units. At 15 minutes data wasonly collected for the ticagrelor+TICA0212/MEDI2452 group. (B)Percentage reversal induced by TICA0212/MEDI2452, mean data (n=4)±SEM

FIG. 12A-B shows the reversal of ticagrelor induced bleeding. (A)Individual data for total blood loss and (B) for total bleeding time.Vehicle control (▪), ticagrelor alone (●) andticagrelor+TICA0212/MEDI2452 (□). Bar represents mean data (n=12).

DETAILED DESCRIPTION

Before continuing to describe the present disclosure in further detail,it is to be understood that this disclosure is not limited to specificcompositions or process steps, as such may vary. It must be noted that,as used in this specification and the appended claims, the singular form“a”, “an” and “the” include plural referents unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisinvention.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The numbering of amino acids in the variable domain, complementaritydetermining region (CDRs) and framework regions (FR), of an antibodyfollow, unless otherwise indicated, the Kabat definition as set forth inKabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insertion (residue 52a according to Kabat) after residue 52 of H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence. Maximal alignment of framework residues frequentlyrequires the insertion of “spacer” residues in the numbering system, tobe used for the Fv region. In addition, the identity of certainindividual residues at any given Kabat site number may vary fromantibody chain to antibody chain due to interspecies or allelicdivergence.

As used herein, the terms “antibody” and “antibodies”, also known asimmunoglobulins, encompass monoclonal antibodies (including full-lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodiesformed from at least two different epitope binding fragments (e.g.,multispecific antibodies, e.g., PCT publication WO2009018386, PCTApplication No. PCT/US2012/045229, incorporated herein by reference inits entirety), biMabs, human antibodies, humanized antibodies, camelisedantibodies, single-chain Fvs (scFv), single-chain antibodies, singledomain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments,antibody fragments that exhibit the desired biological activity (e.g.the antigen binding portion), disulfide-linked Fvs (dsFv), andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), intrabodies, and epitope-bindingfragments of any of the above. In particular embodiments providedherein, antibodies relate to active binding fragments of an antibody,i.e., molecules that contain at least one antigen-binding site such as,for example scFv and Fab. Antibodies also include peptide fusions withantibodies or portions thereof such as a protein fused to an Fc domain.Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM,IgD, IgA and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or allotype (e.g., Gm, e.g., G1m(f, z, a or x), G2m(n), G3m(g, b,or c), Am, Em, and Km(1, 2 or 3)). Antibodies may be derived from anymammal, including, but not limited to, humans, monkeys, pigs, horses,rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g.chickens).

As used herein, C₁-C₆ alkyl refers to straight chain and branded alkylshaving one to six carbon atoms, and includes, methyl, ethyl, propyl,n-butyl, iso-butyl, pentyl, isopentyl, neopentyl, and hexyl.

As used herein, C₁-C₆ alkoxy refers to an alkyl, as noted above, havingan oxygen in the group. In some embodiments, the oxygen atom is locatedat the position that attaches the substituent group to the corestructure (i.e., ring structure).

As used herein, C₁-C₆ alkylthio refers to an alkyl, as noted above,having a sulfur in the group. In some embodiments, the sulfur atom islocated at the position that attaches the substituent group to the corestructure (i.e., ring structure).

As used herein, C₁-C₆ alkanol refers to an alkyl, as noted above, havinga hydroxyl group at the terminal end of the substituent structure.

As used herein, C₃-C₆ cycloalkyl refers to cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl.

As used herein, “substituted” C₃-C₆ cycloalkyl and C₁-C₆ alkyl refer tothe alkyl and cycloalkyl groups discussed above which are substituted onat least one carbon atom with an aryl group that is also substitutedwith 1-3 halogen atoms.

As used herein, “ticagrelor” refers to the reversible P2Y₁₂ inhibitor((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol)and having the chemical structure:

As used herein, “ticagrelor active metabolite” or “TAM” refers to themajor active metabolite of ticagrelor, also referred to as AR-C124910XX,a reversible P2Y₁₂ inhibitor and having the chemical structure:

As used herein, “ticagrelor inactive metabolite” or “TIM” refers to aninactive metabolite of ticagrelor, also referred to as AR-C133913XX, andhaving the chemical structure:

Antibodies

In a general sense, the disclosure provides novel antibodies that bind acyclopentyltriazolopyrimidine compound of the Formula (Ia):

-   -   wherein    -   R₁ is selected from the group consisting of C₁-C₆ alkoxy and        C₁-C₆ alkylthio;    -   R₂ is selected from the group consisting of H, C₁-C₆ alkyl,        substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and substituted C₃-C₆        cycloalkyl; and    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl,        C₁-C₆ alkoxy, and C₁-C₆ alkanol.

In particular embodiments, the antibodies specifically bind a compoundselected from the from the group consisting of

In particular aspects, the disclosure provides an antibody that binds toticagrelor and TAM with any one or more of the following featuresincluding high binding specificity, high binding affinity, rapid time toonset, and rapid time to offset (e.g., allowing for the optionalcontinuation of or co-administration of therapy comprising ticagrelor).

In some embodiments, the antibody binds to ticagrelor and neutralizesthe anti-platelet aggregation activity of ticagrelor and TAM, thusrestoring ADP-induced platelet aggregation in the presence of ticagrelorand TAM.

In some embodiments, the antibody half-life in a subject is about thesame as the half-life of ticagrelor and TAM. In some embodiments theantibody half-life is from about 4-24 hours (e.g., 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). Insome embodiments the antibody half-life is from about 4-12 hours (e.g.,4, 5, 6, 7, 8, 9, 10, 11, or 12 hours).

In some embodiments, the antibody provides for a rapid onset ofactivity. For example, in embodiments the antibody time to onset or thetime to neutralize ticagrelor and TAM mediated platelet inhibition, isfrom about 15-120 minutes, or from about 15-60 minutes. In someembodiments, the time to onset is less than 60 minutes.

In some embodiments the antibody has a PK/PD profile that provides for arapid offset of activity, such that, for example, a subject who has beenadministered the antibody may recommence with the prescribed ticagrelortherapy. In some embodiments, a subject who has received an antibodydisclosed herein (e.g., by i.v. infusion) may receive or restartticagrelor therapy within twenty-four hours following the administrationof the antibody.

As discussed and exemplified in certain embodiments herein, the antibodybinds ticagrelor or a metabolite thereof and does not bind to otherstructurally related compounds, or compounds that may be administeredwith ticagrelor as a cotherapy. For example, suitably, the antibody doesnot inhibit the activity of a compound selected from the groupconsisting of fenofibrate, nilvadipine, cilostazol, bucladesine,regadenoson, cyclothiazide, cyfluthrin, lovastatin, linezolid,simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate,adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosinetriphosphate.

The antibodies described herein can comprise antigen binding fragmentscontaining only select portions of an antibody molecule, such as Fab,F(ab′)₂, Fab′, scFv, di-scFv, sdAb fragments, and may be used asdiagnostic or therapeutic agents. In addition, specific residues in thevariable domains may be altered to improve binding specificity and/orstability of antibodies and antibody fragments. Other residues notdirectly involved in antigen binding have been replaced in order to“humanize” regions of non-human antibodies and reduce immunogenicity ofthe antibody.

In certain aspects, the antibody is a Fab fragment, for example, a Fabfragment of an antibody or a recombinantly produced antigen bindingfragment comprising a variable light chain (VL), a constant light chain(CL), a variable heavy chain (VH), and a constant heavy chain portion(CH1). Optionally, the light and heavy chains of the Fab may beinterconnected via one or more disulfide linkages such as, for example,via a suitable antibody hinge region. As described herein, the Fab bindsto an epitope of a compound of the cyclopentyltriazolopyrimidine classof oral active agents. In some embodiments the Fab binds to ticagreloror a metabolite thereof.

In certain aspects, the Fab may be derived from or based on the sequenceof an antibody, such as a conventional murine, humanized or humanantibody. In certain aspects, the Fab may be derived from or based onone or more scFvs, such as scFvs screened and derived from a library. Insuch embodiments, the Fab derived from or based on the sequence of aconventional antibody or scFv retains one or more functional activitiesof the conventional antibody (e.g., retains at least 80% or more (80%,85%, 90%, 95%, 97%, 98%, 99% or 100%) of a functional activity). Forexample, in certain aspects, the Fab retains one or more of the affinityfor antigen (e.g., ticagrelor), inhibitory activity, and/or selectivityof the antibody or scFv.

While the Fab fragment may comprise a sequence that binds to an epitopeof a cyclopentyltriazolopyrimidine, in certain embodiments, the Fabbinds to ticagrelor. In some aspects, the Fab binds to the activemetabolite of ticagrelor. In certain aspects, the Fab may bind to bothticagrelor and the active metabolite of ticagrelor.

In some embodiments the Fab may comprise a combination of CDR regionsfrom different antibodies that bind to ticagrelor or the activemetabolite thereof.

In certain aspects, the Fab comprises a light chain portion (VL)comprising the amino acid sequence set forth in any of SEQ ID NO:7, SEQID NO: 17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQID NO:67, and SEQ ID NO:77. In further embodiments the Fab comprises alight chain portion comprising the amino acid sequence set forth in anyof SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In certain aspects, theFab comprises a heavy chain portion (VH) comprising the amino acid asset forth in any of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ IDNO:32, SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:62, and SEQ ID NO:72. Infurther embodiments, the Fab comprises a heavy chain portion comprisingthe amino acid sequence set forth in any SEQ ID NO:52, SEQ ID NO:62, andSEQ ID NO:72. In certain aspects, the Fab is encoded by a nucleotidesequence encoding the light chain portion (VL) and a nucleotide sequenceencoding the heavy chain portion (VH), for example, a nucleotidesequence comprising the nucleic acid sequence set forth in SEQ ID NO: 1,SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51,SEQ ID NO:61, or SEQ ID NO:71; and a nucleotide sequence comprising thenucleic acid sequence as set forth in SEQ ID NO:6, SEQ ID NO: 16, SEQ IDNO:16, SEQ ID NO:16, SEQ ID NO:16, SEQ ID NO:16, SEQ ID NO:16, or SEQ IDNO:76.

In certain aspects, the antibody may be an scFv. It is understood thatan scFv encompasses a polypeptide chain comprising a variable heavychain domain (VH) linked to a variable light chain domain (VL) via aflexible polypeptide linker. In some aspects the polypeptide linkerbetween VH and VL comprises a protease cleavage site. The VH and VLdomains of the scFv may be derived from the same or from differentantibodies. In some aspects, a VH or VL of the scFv may comprise one ormore CDRs which bind to a target of interest, while the remainder of theVH or VL domain is derived from a different antibody or is synthetic. Insome aspects, the scFv comprises at least one CDR of an antibody, e.g.,an antibody with binding activity to ticagrelor or a metabolite thereof.In some aspects, the scFv comprises at least two CDRs of a givenantibody. In some aspects, the scFv comprises at least three CDRs of agiven antibody. In some aspects, the scFv comprises at least four CDRsof a given antibody. In some aspects, the scFv comprises at least fiveCDRs of a given antibody. In some aspects, the scFv comprises at leastsix CDRs of a given antibody.

Several methodologies can be used alone or in combination to improve thestability of a scFv molecule. One methodology that can be used, alone orin combination with one or more of the other methodologies, isengineering the length and/or composition of the linker connecting thescFv domains to stabilize the scFv portion.

Another potential methodology that can be used, alone or in combinationwith one or more of the other methodologies described herein, is byintroducing at least two amino acid substitutions (also referred to asmodifications or mutations) into the VH and/or VL domains of the scFv soas to promote disulfide bond formation (see for example Brinkmann etal., 1993, PNAS, 90:7538-42; Zhu et al., 1997, Prot. Sci. 6:781-8;Reiter et al., 1994, Biochem. 33:5451-9; Reiter et al., 1996, Nature 14:1239-45; Luo et al., 1995, J. Biochem. 118:825-31; Young et al., 1995,FEBS Let. 377:135-9; Glockshuber et al., 1990, Biochem. 29:1362-7).

In certain aspects, one mutation is introduced into each of the VH andVL domains of the scFv to promote interchain disulfide bond formationbetween the VH and VL domains upon expression of a scFv. In anotheraspect, the two mutations are introduced in the same domain of thechain. In certain aspect, the two mutations are introduced in differentchains. In certain aspects, multiple pairs of two mutations areintroduced to promote formation of multiple disulphide bonds. In certainaspects, a cysteine is introduced to promote the disulphide bondformation. Exemplary amino acids that may be mutated to cysteine includeamino acids 43, 44, 45, 46, 47, 103, 104, 105, and 106 of VH2 and aminoacids 42, 43, 44, 45, 46, 98, 99, 100, and 101 of VL2. The foregoingnumbering is based on Kabat numbering identifying the position relativeonly to the VH2 and VL2 of the scFv (and not relative to the position ofthe amino acid in a full length sequence of an antibody). Exemplarycombinations of amino acid positions which may be mutated to cysteineresidues include: VH44-VL100, VH105-VL43, VH105-VL42, VH44-VL101,VH106-VL43, VH104-VL43, VH44-VL99, VH45-VL98, VH46-VL98, VH103-VL43,VH103-VL44, and VH103-VL45. In some aspects, amino acid 44 of VH andamino acid 100 of VL are mutated to cysteines.

A further potential methodology that can be used, alone or incombination with one or more of the other methodologies describedherein, is selecting the order of the domains of the scFv. In certainaspects, the orientation of the VH domain relative to the VL domain isoptimized for stability. In certain aspects, the scFv is in theVH-linker-VL orientation. In certain aspects, the scFv is in theVL-linker-VH orientation.

An additional methodology that can be used, alone or in combination withone or more of the methodologies described herein, is by introducing oneor more stabilizing mutations by mutating one or more surface residuesof the scFv. In some aspects, one, two, three, four, five, six, or morethan six residues are mutated in one or both of the VH and/or VL domainof the scFv. In certain aspects, changes are made in only the VH domainof the scFv. In certain aspects, changes are made in only the VL domainof the scFv. In certain aspects, changes are made in both the VH and VLdomains of the scFv. The same number of changes may be made in eachdomain or a different number of changes may be made in each domain. Incertain aspects, one or more of the changes is a conservative amino acidsubstitution from the residue present in the unmodified, parent scFv. Inother aspects, one or more of the changes is a non-conservative aminoacid substitution from the residue present in the unmodified, parentscFv. When multiple substitutions are made, either in one or both of theVH or VL domains of the scFv, each substitution is independently aconservative or a non-conservative substitution. In certain aspects, allof the substitutions are conservative substitutions. In certain aspects,all of the substitutions are non-conservative. In certain aspects, atleast one of the substitutions is conservative. In certain aspects, atleast one or the substitutions is non-conservative.

Yet a further methodology that can be used, alone or in combination withone or more of the additional methodologies described herein, is byintroducing one or more substitutions by mutating one or more residuespresent in the VH and/or VL domain of the scFv to match the mostfrequent residue at said particular position of a consensus sequence ofVH and/or VL domain of known, screened, and/or identified antibodies. Incertain aspects, substitutions are introduced at one, two, three, four,five, six, or more than six positions in one or both of the VH domainand/or the VL domain of the scFv. The same number of changes may be madein each domain or a different number of changes may be made in eachdomain. In certain aspects, one or more of the changes in sequence matchthat of a given consensus is a conservative amino acid substitution fromthe residue present in the unmodified VH and/or VL sequence. In otheraspects, one or more of the changes represent a non-conservative aminoacid substitution from the residue present in the unmodified VH and/orVL sequence. When multiple substitutions are made, either in one or bothof the VH or VL domain of the scFv, each substitution is independently aconservative or a non-conservative substitution. In certain aspects, allof the substitutions are conservative substitutions. In certain aspects,all of the substitutions are non-conservative substitutions. In certainaspects, at least one of the substitutions is conservative. In certainaspects, at least one or the substitutions is non-conservative.

It should be noted that any of the modifications described as useful formodifying or stabilizing the scFv portion can be applied to modify a Fabportion. For example, the variable domains of a Fab can be modified toimprove stability, antigen binding and the like. Moreover, either theFab or scFv portion can be modified to reduce immunogenicity.

In certain aspects, the antibody may be a scFv that comprises a variablelight chain portion (VL) comprising the amino acid sequence set forth inany of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ IDNO:47, SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In furtherembodiments the scFv comprises a light chain portion comprising theamino acid sequence set forth in any of SEQ ID NO:57, SEQ ID NO:67, andSEQ ID NO:77. In certain aspects, the scFv comprises a heavy chainportion (VH) comprising the amino acid as set forth in any of SEQ IDNO:2, SEQ ID NO: 12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ IDNO:52, SEQ ID NO:62, and SEQ ID NO:72. In further embodiments, the scFvcomprises a heavy chain portion comprising the amino acid sequence setforth in any SEQ ID NO:52, SEQ ID NO:62, and SEQ ID NO:72.

The antibodies disclosed herein may further comprise one or more linkerpolypeptides. The linker may interconnect a heavy chain domain and alight chain domain (scFv) or connect an antibody or antigen bindingfragment thereof to another agent, such as a label, Fc domain, or thelike. Linkers can vary in length and sequence and are generally known inthe art.

The serum half-life of an antibody comprising an Fc region may beincreased by increasing the binding affinity of the Fc region for FcRn.The term “antibody half-life” as used herein means a pharmacokineticproperty of an antibody that is a measure of the mean survival time ofantibody molecules following their administration. Antibody half-lifecan be expressed as the time required to eliminate 50 percent of a knownquantity of immunoglobulin from the patient's body (or other mammal) ora specific compartment thereof, for example, as measured in serum, i.e.,circulating half-life, or in other tissues. Half-life may vary from oneimmunoglobulin or class of immunoglobulin to another. In general, anincrease in antibody half-life results in an increase in mean residencetime (MRT) in circulation for the antibody administered.

The increase in half-life may allow for the reduction in amount of agentgiven to a patient as well as reducing the frequency of administration.To increase the serum half-life of an antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule. Alternatively, antibodies of the disclosure withincreased half-lives may be generated by modifying amino acid residuesidentified as involved in the interaction between the Fc and the FcRnreceptor (see, for examples, U.S. Pat. Nos. 6,821,505 and 7,083,784; andWO 09/058492). In addition, the half-life of antibodies of thedisclosure may be increased by conjugation to PEG or albumin bytechniques widely utilized in the art.

Antibodies falling within the scope of the disclosure may be identifiedby any of the structural and/or functional characteristics identifiedherein. For example, antibodies may be screened for particular bindingfeatures (e.g., K_(off), K_(D), IC₅₀, Specificity to/selectivity forticagrelor and ticagrelor metabolites) using any of the techniquesillustrated herein or that are otherwise known in the art.

Labels, Conjugates and Moieties

Antibodies of the disclosure may be conjugated to labels for thepurposes of diagnostics and other assays wherein the antibodies and/orits target(s) may be detected. Labels include, without limitation, achromophore, a fluorophore, a fluorescent protein, a phosphorescent dye,a tandem dye, a particle, a hapten, an enzyme and a radioisotope.

In certain aspects, the antibodies are conjugated to a fluorophore. Thechoice of the fluorophore attached to the antibody will determine theabsorption and fluorescence emission properties of the conjugatedantibody. Physical properties of a fluorophore label that can be usedfor an antibody and antibody-bound ligands include, but are not limitedto, spectral characteristics (absorption, emission and stokes shift),fluorescence intensity, lifetime, polarization and photo-bleaching rate,or combination thereof. All of these physical properties can be used todistinguish one fluorophore from another, and thereby allow formultiplexed analysis. Other desirable properties of the fluorescentlabel may include cell permeability and low toxicity, for example iflabeling of the antibody is to be performed in a cell or a modelorganism (e.g., a living animal).

In certain aspects, an enzyme is a label and is conjugated to anantibody. Enzymes are desirable labels because amplification of thedetectable signal can be obtained resulting in increased assaysensitivity. The enzyme itself does not produce a detectable responsebut functions to break down a substrate when it is contacted by anappropriate substrate such that the converted substrate produces afluorescent, colorimetric or luminescent signal. Enzymes amplify thedetectable signal because one enzyme on a labeling reagent can result inmultiple substrates being converted to a detectable signal. The enzymesubstrate is selected to yield the preferred measurable product, e.g.colorimetric, fluorescent or chemiluminescence. Such substrates areextensively used in the art and are well known by one skilled in the artand include for example, oxidoreductases such as horseradish peroxidaseand a substrate such as 3,3′-diaminobenzidine (DAB); phosphatase enzymessuch as an acid phosphatase, alkaline and a substrate such as5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases, such asbeta-galactosidase, beta-glucuronidase or beta-glucosidase and asubstrate such as 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside(X-gal); additional enzymes include hydrolases such as cholinesterasesand peptidases, oxidases such as glucose oxidase and cytochromeoxidases, and reductases for which suitable substrates are known.

Enzymes and their appropriate substrates that produce chemiluminescenceare suitable for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful.

In another aspect, haptens such as biotin, are also utilized as labels.Biotin is useful because it can function in an enzyme system to furtheramplify the detectable signal, and it can function as a tag to be usedin affinity chromatography for isolation purposes. For detectionpurposes, an enzyme conjugate that has affinity for biotin is used, suchas avidin-HRP. Subsequently a peroxidase substrate is added to produce adetectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs,pollutants, allergens, affector molecules, growth factors, chemokines,cytokines, lymphokines, amino acids, peptides, chemical intermediates,nucleotides and the like.

In certain aspects, fluorescent proteins may be conjugated to theantibody as a label. Examples of fluorescent proteins include greenfluorescent protein (GFP) and the phycobiliproteins and the derivativesthereof. The fluorescent proteins, especially phycobiliprotein, areparticularly useful for creating tandem dye labeled labeling reagents.These tandem dyes comprise a fluorescent protein and a fluorophore forthe purposes of obtaining a larger stokes shift wherein the emissionspectra is farther shifted from the wavelength of the fluorescentprotein's absorption spectra.

In certain aspects, the label is a radioactive isotope. Examples ofsuitable radioactive materials include, but are not limited to, iodine(¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹¹In, ¹¹²In, ¹¹³mIn, ¹¹⁵mIn), technetium (⁹⁹Tc, ⁹⁹mTc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³⁵Xe), fluorine (¹⁸F), ¹⁵³SM, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh and ⁹⁷Ru.

In some aspects, drugs may be conjugated to the antibody. For example,an antibody comprising an scFv may be conjugated to a drug for thetreatment of a cardiovascular disease and/or acute coronary syndromes.

In certain features, drugs and other molecules may be targeted to anantibody via site-specific conjugation. For example, the antibody maycomprise cysteine engineered domains (including cysteine(s) engineeredinto a binding unit and/or Fc domain), which result in free thiol groupsfor conjugation reactions. In certain aspects, an antibody is engineeredto incorporate specific conjugation sites.

Nucleic Acid Molecules Encoding Antibodies

The present disclosure provides nucleic acid molecules that encodeantibodies or antigen-binding fragments thereof. One aspect of thedisclosure provides nucleic acid molecules encoding any of theantibodies specifically described herein. A nucleic acid molecule mayencode a variable region of a heavy chain and/or light chain of theantibody.

In some aspects, the antibody is a Fab or scFv, wherein the nucleic acidportion encoding the Fab or scFv comprises a nucleotide sequenceencoding a VL domain and a nucleotide sequence encoding a VH, andwherein the nucleotide sequence encoding the VL domain is optionallylinked to the nucleotide sequence encoding the VH domain via anucleotide sequence encoding a flexible polypeptide linker.

A further aspect provides a host cell transformed with any of thenucleic acid molecules as described herein. In another aspect of thedisclosure there is provided a host cell comprising a vector comprisingnucleic acid molecules as described herein. In one aspect the host cellmay comprise more than one vector.

The disclosure contemplates nucleic acid molecules encoding any antibodyof the disclosure, as well as either the light or heavy chain of anantibody. For example, the disclosure contemplates a nucleic acidmolecule comprising a nucleotide sequence encoding one or more of SEQ IDNO:2, SEQ ID NO: 12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ IDNO:52, SEQ ID NO:62, SEQ ID NO:72, SEQ ID NO:7, SEQ ID NO:17, SEQ IDNO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, and SEQID NO:77. The disclosure further contemplates nucleic acid moleculesencoding any antibody of the disclosure further comprising additionalregions (e.g., Fc or modified Fc). In some embodiments the nucleic acidmolecules may be selected from one or more of SEQ ID NO: 1, SEQ ID NO:6,SEQ ID NO:11, SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:56,SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:71, or SEQ ID NO:76. In furtherembodiments, the disclosure provides a vector comprising a nucleic acidmolecule selected from one or more of SEQ ID NO: 1, SEQ ID NO:6, SEQ IDNO:11, SEQ ID NO: 16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, SEQ IDNO:36, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:56, SEQ IDNO:61, SEQ ID NO:66, SEQ ID NO:71, or SEQ ID NO:76.

Methods for Producing Antibodies, Fabs, and scFvs

The disclosure provides methods for producing the antibodies andfragments thereof that are described herein. In some aspects,antigen-binding fragments of antibodies which recognize ticagrelor andthe specific epitopes of ticagrelor and/or TAM disclosed herein may begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)₂ fragments may be produced from antibodies byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). Further, the antibodies including scFvs and Fabs, asdescribed herein, can be generated using various phage display methodsknown in the art.

Generally, in phage display methods, functional antibody domains aredisplayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of lymphoid tissues). The DNAencoding the VH and VL domains are recombined together with an scFvlinker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are may be filamentous phage including fdand M13 and the VH and VL domains may be recombinantly fused to eitherthe phage gene III or gene VIII. Phage expressing an antigen bindingdomain that binds to ticagrelor and/or TAM can be selected or identifiedwith antigen, e.g., using labeled antigen or antigen bound or capturedto a solid surface or bead. Similarly, binding domains that bind toantigens/haptens in addition to or other than to ticagrelor and/or TAMcan be identified for deselection. Examples of phage display methodsthat can be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough etal., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCTapplication No. PCT/GB91/O1 134; PCT publication Nos. WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment (scFvs and Fabs), and expressed in anydesired host, including mammalian cells, insect cells, plant cells,yeast, and bacteria, e.g., as described below. Techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., 1992, BioTechniques12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043 (said references incorporated by referencein their entireties).

In certain aspects, the nucleic acids disclosed herein may be operablylinked to one or more regulatory nucleotide sequences in an expressionconstruct. The nucleic acid sequences encoding the antibody light andheavy chains can be cloned in the same expression vector in anyorientation (e.g., light chain in front of the heavy chain or viceversa) or can be cloned in two different vectors. If expression iscarried out using one vector, the two coding genes can have their owngenetic elements (e.g., promoter, RBS, leader, stop, polyA, ect) or theycan be cloned with one single set of genetic elements, but connectedwith a cistron element. Regulatory nucleotide sequences will generallybe appropriate for a host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome.

In certain aspects, the expression vector contains a selectable markergene to allow the selection of transformed host cells. Selectable markergenes are well known in the art and will vary with the host cell used.In certain aspects, this disclosure relates to an expression vectorcomprising a nucleotide sequence encoding a polypeptide and operablylinked to at least one regulatory sequence. Regulatory sequences areart-recognized and are selected to direct expression of the encodedpolypeptide. Accordingly, the term regulatory sequence includespromoters, enhancers, and other expression control elements. Exemplary,non-limiting regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego,Calif. (1990). It should be understood that the design of the expressionvector may depend on such factors as the choice of the host cell to betransformed and/or the type of protein desired to be expressed.Moreover, the vector's copy number, the ability to control that copynumber and the expression of any other protein encoded by the vector,such as antibiotic markers, should also be considered.

The methods for producing an antibody of the disclosure may include, forexample, a host cell transfected with one or more than one expressionvectors encoding an antibody (e.g., a single vector encoding the heavyand the light chain or variable regions thereof, or two vectors, oneencoding the heavy chain and one encoding the light chain or variableregions thereof) can be cultured under appropriate conditions to allowexpression of the antibody to occur. The antibody may be secreted andisolated from a mixture of cells and medium containing the antibody.Alternatively, the antibody may be retained in the cytoplasm or in amembrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The antibody can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins,antibodies, and antigen binding antibody fragments thereof, includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification. Incertain aspects, the antibody is made as an antigen binding fragment ofan antibody that comprises the heavy and light chain variable regions,which may increase solubility and facilitate purification.

A recombinant nucleic acid can be produced by ligating the cloned gene,or a portion thereof, into a vector suitable for expression in eitherprokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian),or both. Expression vehicles for production of a recombinant polypeptideinclude plasmids and other vectors. For instance, suitable vectorsinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli. Incertain aspects, mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular CloningALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press, 1989) Chapters 16 and 17. In someinstances, it may be desirable to express the recombinant polypeptide bythe use of a baculovirus expression system. Examples of such baculovirusexpression systems include pVL-derived vectors (such as pVL1392, pVL1393and pVL941), pAcUW-derived vectors (such as pAcUWI), andpBlueBac-derived vectors (such as the 3-gal containing pBlueBac III).

Techniques for making fusion genes are well known. Essentially, thejoining of various nucleic acid fragments coding for differentpolypeptide/antibody sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another aspect, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive nucleic acid fragments which can subsequently beannealed to generate a chimeric gene sequence (see, for example, CurrentProtocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons:1992).

In some aspects, an expression vector expressing any of the nucleicacids described herein may be used to express the antibody in a hostcell. For example, an antibody may be expressed in bacterial cells suchas E. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Once the expression vector is transferred to a host cell by conventionaltechniques, the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the disclosure includes hostcells containing a polynucleotide encoding an antibody or fragmentsthereof, operably linked to a heterologous promoter. In certain aspects,both the heavy chain and the light chain and/or heavy and light chainvariable regions may be co-expressed (from the same or differentvectors) in the host cell for expression of the entire antibody. Incertain aspects, both the heavy and light chains of the antibody areexpressed from a single promoter. In certain aspects, the heavy andlight chains of the antibody are expressed from multiple promoters. Incertain aspects, the heavy and light chains of the antibody are encodedon a single vector. In certain aspects, the heavy and light chains ofthe antibody are encoded on multiple vectors.

Mammalian cell lines available as hosts for expression of recombinantantibodies are well known in the art and include many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney293 cells, and a number of other cell lines. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody or portion thereofexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO(a murine myeloma cell line that does not endogenously produce anyfunctional immunoglobulin chains), SP20, CRL7030 and HsS78Bst cells. Inone aspect, human cell lines developed by immortalizing humanlymphocytes can be used to recombinantly produce monoclonal antibodies.In one aspect, the human cell line PER.C6. (Crucell, Netherlands) can beused to recombinantly produce monoclonal antibodies.

Additional cell lines which may be used as hosts for expression ofrecombinant antibodies include, but are not limited to, insect cells(e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5bl-4) or yeast cells (e.g. S.cerevisiae, Pichia, U.S. Pat. No. 7,326,681; etc), plants cells(US20080066200); and chicken cells (WO2008142124).

In certain aspects, an antibody of the disclosure is stably expressed ina cell line. Stable expression can be used for long-term, high-yieldproduction of recombinant proteins, antibodies and antigen-bindingfragments thereof. For example, cell lines which stably express theantibody molecule may be generated. Host cells can be transformed withan appropriately engineered vector comprising expression controlelements (e.g., promoter, enhancer, transcription terminators,polyadenylation sites, etc.), and a selectable marker gene. Followingthe introduction of the foreign DNA, cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells that stably integrated theplasmid into their chromosomes to grow and form foci which in turn canbe cloned and expanded into cell lines. Methods for producing stablecell lines with a high yield are well known in the art and reagents aregenerally available commercially.

In certain aspects, an antibody of the disclosure is transientlyexpressed in a cell line. Transient transfection is a process in whichthe nucleic acid introduced into a cell does not integrate into thegenome or chromosomal DNA of that cell. It is in fact maintained as anextrachromosomal element, e.g. as an episome, in the cell. Transcriptionprocesses of the nucleic acid of the episome are not affected and aprotein encoded by the nucleic acid of the episome is produced.

The cell line, either stable or transiently transfected, is maintainedin cell culture medium and conditions well known in the art resulting inthe expression and production of monoclonal antibodies. In certainaspects, the mammalian cell culture media is based on commerciallyavailable media formulations, including, for example, DMEM or Ham's F12.In other aspects, the cell culture media is modified to supportincreases in both cell growth and biologic protein expression. As usedherein, the terms “cell culture medium,” “culture medium,” and “mediumformulation” refer to a nutritive solution for the maintenance, growth,propagation, or expansion of cells in an artificial in vitro environmentoutside of a multicellular organism or tissue. Cell culture medium maybe optimized for a specific cell culture use, including, for example,cell culture growth medium which is formulated to promote cellulargrowth, or cell culture production medium which is formulated to promoterecombinant protein production. The terms nutrient, ingredient, andcomponent are used interchangeably herein to refer to the constituentsthat make up a cell culture medium.

Once a molecule has been produced, it may be purified by any methodknown in the art for purification of an immunoglobulin molecule orfragments thereof, for example, by chromatography (e.g., ion exchange,affinity, particularly by affinity for the specific antigens Protein Aor Protein G, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins, antibodies, and/or antibody fragments.Further, the molecules of the present disclosure or fragments thereofmay be fused to heterologous polypeptide sequences (referred to hereinas “tags” such as a histidine tag) described herein or otherwise knownin the art to facilitate purification.

When using recombinant techniques, the molecule can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the molecule is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio Technology, 10:163-167 (1992) describe a procedure forisolating antibodies which are secreted into the periplasmic space of E.coli. Where the molecule is secreted into the medium, supernatants fromsuch expression systems are generally first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. A protease inhibitorsuch as PMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The composition prepared from the cells can be purified using, forexample, hydroxylapatite chromatography, hydrophobic interactionchromatography, ion exchange chromatography, gel electrophoresis,dialysis, and/or affinity chromatography either alone or in combinationwith other purification steps. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain, if present, in the molecule and will be understood by one ofskill in the art. The matrix to which the affinity ligand is attached ismost often agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin, SEPHAROSE chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the molecule to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe molecule of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, and performed at low salt concentrations (e.g.,from about 0-0.25 M salt).

An antibody may be made and purified using, for example, any one orcombination of techniques set forth above and/or in the Examples.Regardless of how an antibody is purified, to confirm functional bindingof the antibody of the disclosure, binding assays may be performed(before and/or after purification). For example, dual ELISA assays maybe used. In some aspects, a first antigen (e.g., ticagrelor or acompetitor thereof) is coated on a well, and binding to this antigenimmobilizes the antibody preparing it for detection.

Pharmaceutical Formulations

In certain aspects, the disclosure provides pharmaceutical compositions.Such pharmaceutical compositions may be compositions comprising anucleic acid molecule that encodes an antibody. Such pharmaceuticalcompositions may also be compositions comprising an antibody, or acombination of antibodies, and a pharmaceutically acceptable excipient.In certain aspects, the pharmaceutical compositions of the disclosureare used as a medicament.

In certain aspects, an antibody or a combination of antibodies (ornucleic acid molecules encoding an antibody or a combination ofantibodies) may be formulated with a pharmaceutically acceptablecarrier, excipient or stabilizer, as pharmaceutical compositions. Incertain aspects, such pharmaceutical compositions are suitable foradministration to a human or non-human animal via any one or more routeof administration using methods known in the art. As will be appreciatedby the skilled artisan, the route and/or mode of administration willvary depending upon the desired results. The term “pharmaceuticallyacceptable carrier” means one or more non-toxic materials that do notinterfere with the effectiveness of the biological activity of theactive ingredients. Such preparations may routinely contain salts,buffering agents, preservatives, compatible carriers, and optionallyother therapeutic agents. Such pharmaceutically acceptable preparationsmay also contain compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration into ahuman. Other contemplated carriers, excipients, and/or additives, whichmay be utilized in the formulations described herein include, forexample, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, lipids, protein excipients such asserum albumin, gelatin, casein, salt-forming counterions such as sodiumand the like. These and additional known pharmaceutical carriers,excipients and/or additives suitable for use in the formulationsdescribed herein are known in the art, e.g., as listed in “Remington:The Science & Practice of Pharmacy”, 21^(st) ed., Lippincott Williams &Wilkins, (2005), and in the “Physician's Desk Reference”, 60^(th) ed.,Medical Economics, Montvale, N.J. (2005). Pharmaceutically acceptablecarriers can be selected that are suitable for the mode ofadministration, solubility and/or stability desired or required.

The formulations described herein comprise active agents (e.g.,antibodies or antibody fragments such as Fab or scFv) in a concentrationresulting in a w/v appropriate for a desired dose. In certain aspects,the active agent is present in a formulation at a concentration of about1 mg/ml to about 200 mg/ml, about 1 mg/ml to about 100 mg/ml, about 1mg/ml to about 50 mg/ml, or about 1 mg/ml to about 25 mg/ml. In certainaspects, the active agent is present at a concentration of about 25mg/ml. In certain aspects, the concentration of the active agent in aformulation may vary from about 0.1 to about 100 weight %. In certainaspects, the concentration of the active agent is in the range of 0.003to 1.0 molar.

In one aspect, the formulations of the disclosure are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances (glycoproteins) from the outer membrane ofbacteria and other microorganisms. Both of these substances can causefever, hypotension and shock if administered to humans. Due to thepotential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Incertain specific aspects, the endotoxin and pyrogen levels in thecomposition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than0.001 EU/mg.

When used for in vivo administration, the formulations of the disclosureshould be sterile. The formulations of the disclosure may be sterilizedby various sterilization methods, including sterile filtration,radiation, etc. In one aspect, the formulation is filter-sterilized witha presterilized 0.22-micron filter. Sterile compositions for injectioncan be formulated according to conventional pharmaceutical practice asdescribed in “Remington: The Science & Practice of Pharmacy”, 21′ ed.,Lippincott Williams & Wilkins, (2005).

Therapeutic compositions of the present disclosure can be formulated forparticular routes of administration, such as oral, nasal, pulmonary,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The phrases “parenteral administration” and“administered parenterally” as used herein refer to modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrastemal injection and infusion. Formulations of the presentdisclosure which are suitable for topical or transdermal administrationinclude powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. The antibodies may be mixed understerile conditions with a pharmaceutically acceptable carrier, and withany preservatives, buffers, or propellants which may be required (U.S.Pat. Nos. 7,378,110; 7,258,873; 7,135,180; US Publication No.2004-0042972; and 2004-0042971).

The formulations may conveniently be presented in unit dosage form andmay be prepared by any method known in the art of pharmacy. Actualdosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient (e.g., “atherapeutically effective amount”). The selected dosage level willdepend upon a variety of pharmacokinetic factors including the activityof the particular compositions employed, the route of administration,the time of administration, the rate of excretion of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompositions employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts. Suitable dosages may range from about0.0001 to about 100 mg/kg of body weight or greater, for example about0.1, 1, 10, or 50 mg/kg of body weight, with about 1 to about 10 mg/kgof body weight being suitable.

Note that the disclosure similarly contemplates that formulationssuitable for diagnostic and research use may also be made. Theconcentration of active agent in such formulations, as well as thepresence or absence of excipients and/or pyrogens can be selected basedon the particular application and intended use.

Uses

The antibodies disclosed herein are useful in therapeutic methods,including combination therapy, for neutralizing the activity ofinhibitors of platelet activation, aggregation and degranulation,promoters of platelet disaggregation, and anti-thrombotic agents. Thus,the antibodies described herein, find use in a number of applicationsthat relate to the administration of ticagrelor (including companionmethods) and are suitably useful for neutralizing the effect ofticagrelor and/or one or more metabolites of ticagrelor. In suchmethods, the antibody can reduce, neutralize, eliminate, or otherwiseinhibit the activity of ticagrelor, optionally reversibly, and treat orprevent any number of effects, disorders and/or symptoms associated withticagrelor administration and/or arising from therapy comprisingticagrelor.

The antibodies may be administered to patients who are being treated, orwho are in need of treatment or prophylaxis for indications that aretreatable and/or indicated for ticagrelor (BRILINTA), including forexample, unstable angina, primary arterial thrombotic complications ofatherosclerosis such as thrombotic or embolic stroke, transientischaemic attacks, peripheral vascular disease, myocardial infarctionwith or without thrombolysis, arterial complications due tointerventions in atherosclerotic disease such as angioplasty, includingcoronary angioplasty (PTCA), endarterectomy, stent placement, coronaryand other vascular graft surgery, thrombotic complications of surgicalor mechanical damage such as tissue salvage following accidental orsurgical trauma, reconstructive surgery including skin and muscle flaps,conditions with a diffuse thrombotic/platelet consumption component suchas disseminated intravascular coagulation, thrombotic thrombocytopaenicpurpura, haemolytic uraemic syndrome, thrombotic complications ofsepticaemia, adult respiratory distress syndrome, anti-phospholipidsyndrome, heparin-induced thrombocytopaenia and pre-eclampsia/eclampsia,or venous thrombosis such as deep vein thrombosis, venoocclusivedisease, haematological conditions such as myeloproliferative disease,including thrombocythaemia, sickle cell disease; or in the prevention ofmechanically-induced platelet activation in vivo, such ascardio-pulmonary bypass and extracorporeal membrane oxygenation(prevention of microthromboembolism), mechanically-induced plateletactivation in vitro, such as use in the preservation of blood products,e.g. platelet concentrates, or shunt occlusion such as in renal dialysisand plasmapheresis, thrombosis secondary to vascular damage/inflammationsuch as vasculitis, arteritis, glomerulonephritis, inflammatory boweldisease and organ graft rejection, conditions such as migraine.

In some embodiments the antibodies disclosed herein may be administeredto a patient who is receiving or who has received treatment comprisingticagrelor, and who is in need of treatment, or will be in need oftreatment, for bleeding or potential bleeding associated with coronaryartery bypass grafting (CABG), cardiothoracic surgery, mediastinalre-exploration, post-operative stroke, mechanical ventilation, prolongedstay in an intensive care unit, urgent non-cardiac surgery (for example,neurological or ophthalmological surgery, spinal surgery, intracranialsurgery, orbital surgery, orthopedic surgery, nephrectomy,hemicolectomy, and the like. As such, the methods provided herein mayencompass administration of the antibody as a co-therapeutic withticagrelor (simultaneously), or within a period of time followingticagrelor administration (e.g., minutes, hours, or days). For example,in some embodiments, the method may include administration of theantibody to a patient who has been administered ticagrelor within 10-120minutes of ticagrelor administration. In some embodiments, the methodmay include administration of the antibody to a patient who has beenadministered ticagrelor within 1-48 hours of ticagrelor administration.In some embodiments, the antibody is administered to a subject who hasbeen administered ticagrelor within an amount of time that would notallow for the metabolism and elimination of ticagrelor and/or itsmetabolites from the subject.

In some embodiments, the disclosure provides a method of inhibiting theeffect of ticagrelor, or an active metabolite thereof, on the (P2Y₁₂)receptor in a patient.

In some embodiments, the disclosure provides a method of inhibiting thebinding of ticagrelor, or an active metabolite thereof, to a P2Y₁₂receptor in a patient.

In some embodiments, the disclosure provides a method of activatingADP-induced platelet aggregation in a patient who has been administeredticagrelor.

The antibodies of the disclosure, such as those exemplified in theExamples, may also be used for diagnostic purposes. For example, one ormore target agents (ticagrelor or metabolites thereof) may be detectedin tissues or cells of a subject in order to determine or screen forcirculating amounts of ticagrelor in a subject. A diagnostic kit maycomprise one or more antibody, and a detection system for indicating thereaction of the antibody with ticagrelor or metabolites thereof, if anyare present.

Thus, the disclosure contemplates numerous uses for the antibodies,including therapeutic, diagnostic, and research uses. Diagnostic andresearch uses may be in vivo or ex vivo.

Kits

Another aspect of the present disclosure is a kit. In one aspect, a kitcomprises any of the compositions or pharmaceutical compositions of anucleic acid, antibody, expression vector, or host cell described above,and instructions or a label directing appropriate use or administration.Optionally, a kit may also include one or more containers and/or asyringe or other device to facilitate delivery or use. The disclosurecontemplates that all or any subset of the components for conductingresearch assays, diagnostic assays and/or for administeringtherapeutically effective amounts may be enclosed in the kit. Similarly,the kit may include instructions for making an antibody by, for exampleculturing a host cell that expresses a nucleic acid that encodes anantibody of the disclosure under suitable conditions. By way ofadditional example, a kit for therapeutic administration of an antibodyof the disclosure may comprise a solution containing a pharmaceuticalformulation of the antibody, or a lyophilized preparation of theantibody, and instructions for administering the composition to apatient in need thereof and/or for reconstituting the lyophilizedproduct. In certain embodiments the kit will further comprise ticagrelorin a formulation appropriate for administration to a subject (e.g.,BRILINTA™, BRILIQUE™). In such embodiments, the kit may further compriseinstructions for the administration of both the antibody and ticagrelorformulation to a patient in need of treatment with the antibody, withticagrelor, or with both the antibody and ticagrelor.

The present disclosure also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In thecase of dosage forms suitable for parenteral administration the activeingredient, e.g., an above-described antibody, and/or a ticagrelorformulation, is sterile and suitable for administration as a particulatefree solution. In certain aspects, the formulation is suitable for aninjectable route of administration. In some embodiments theadministration is subcutaneous. In some embodiments the administrationis intravenous administration. Thus, routes of administration includinginjection or infusion to a human or animal are contemplated.

In a specific aspect, the formulations of the disclosure are formulatedin single dose vials as a sterile liquid. Exemplary containers include,but are not limited to, vials, bottles, pre-filled syringes, IV bags,blister packs (comprising one or more pills). Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human diagnosis and/oradministration.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the disclosure include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, etc., and other monitoring information.

A kit for diagnostic assays may comprise a solution containing anantibody or a lyophilized preparation of an antibody of the disclosure,wherein the antibody binds specifically to ticagrelor and/or ametabolite thereof, as well as reagents for detecting such an antibody.The antibody may be labeled according to methods known in the art anddescribed herein, including but not limited to labels such as smallmolecule fluorescent tags, proteins such as biotin, GFP or otherfluorescent proteins, or epitope sequences such as his or myc.Similarly, primary antibodies used for detecting an antibody may beincluded in the kit. Primary antibodies may be directed to sequences onthe antibody or to labels, tags, or epitopes with which the antibody islabeled. Primary antibodies may, in turn, be labeled for detection, or,if further amplification of the signal is desired, the primaryantibodies may be detected by secondary antibodies, which may also beincluded in the kit.

Kits for research use are also contemplated. Such kits may, for example,resemble kits intended for diagnostic or therapeutic uses but furtherinclude a label specifying that the kit and its use is restricted toresearch purposes only.

EXAMPLES List of Abbreviations

Abbreviation Explanation ACN acetonitrile br broad BSA Bovine serumalbumin CV column volume d doublet dd double doublet DCM dichloromethaneDMF N,N-dimethylformamide DMSO dimethylsulphoxide DPBS Dulbecco'sphosphate buffered saline EDC1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) EtOAc ethylacetate FAformic acid HOAc acetic acid HPLC high-performance liquid chromatographyHRMS high resolution mass spectrometry HTS high throughput screen HTRF ®homogeneous time resolved fluorescence HYFLO ® filter aid, fluxcalcined, treated with sodium carbonate Hz Hertz J coupling constant LCliquid chromatography m multiplet MS mass spectra NMR nuclear magneticresonance OAc acetate Pd/C Palladium on charcoal pM picomolar PK/PDPharmacokinetic/Pharmacodynamic KF Potassium fluoride q quartet r.t.room temperature s singlet sat. saturated scFv single chain fragmentvariable t triplet TFA trifluoroacetic acid TEA triethylamine TBMEtert-butyl methyl ether THF tetrahydrofuran TIM ticagrelor inactivemetabolite TLC thin layer chomatography TR-FRET Time resolvedfluorescence resonance energy transfer

Example 1: Preparation and Characterization of Haptens

This example describes methods of synthesis, optimization, isolation,and characterization of several haptens that were used to generateexemplary antibodies as described herein. The haptens includeticagrelor, ticagrelor metabolites (TAM and TIM), biotinylatedticagrelor, and biotinylated adenosine (see, e.g., FIG. 2 for chemicalhapten structures in non-biotinylated forms). Ticagrelor was synthesizedas described in international patent publication WO 2000/034283 (Guile,et al., 2000) and TAM was synthesized as described in internationalpatent publication WO 1999/005143 (Guile, et al., 1999), eachincorporated by reference in their entirety.

Straight phase chromatography was performed using Biotage silica gel40S, 40M, 12i or Merck silica gel 60 (0.063-0.200 mm).Flash-chromatography was performed using either standard glass- orplastic-columns or on a Biotage Horizon system. Chemical shifts aregiven in ppm with the solvent as internal standard. Protones onheteroatoms such as NH and OH protons are only reported when detected inNMR and can therefore be missing.

Example 1.1: Biotinylated TicagrelorN-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,3-dihydroxycyclopentyl)oxy)ethyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(1.1)

(i) Preparation of2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethylmethanesulfonate (1.a)

Methanesulfonyl chloride (0.086 mL, 1.10 mmol) was added dropwise to asolution of2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethanol(See Springthorpe, B. et. al. Bioorg. Med. Chem. Lett., 2007, 17,6013-6018) (0.563 g, 1.0 mmol) and TEA (0.209 mL, 1.50 mmol) in DCM (5mL) at 0° C. The mixture was stirred from 0° C. to about 5° C. over 3 h.The reaction mixture was diluted with DCM (30 mL) and washed with water(5 mL). The mixture was dried by passing through a phase separator.Evaporation of the solvent and co-evaporation from toluene gave thetitle compound (1a) (714 mg, 111%) as a yellow thick oil, which was usedas crude without further purification.

¹H NMR (400 MHz, CDCl₃) δ 1.02 (dd, 3H), 1.3-1.47 (m, 5H), 1.55 (s, 3H),1.72 (d, 2H), 2.20 (d, 1H), 2.6-2.71 (m, 2H), 2.97 (s, 3H), 3-3.19 (m,3H), 3.57-3.68 (m, 1H), 3.69-3.79 (m, 1H), 4.02 (td, 1H), 4.13-4.24 (m,2H), 4.78 (dd, 1H), 5.13 (td, 1H), 5.57 (s, 1H), 6.50 (s, 1H), 7.03 (s,1H), 7.07-7.16 (m, 2H).

¹⁹F NMR (376 MHz, CDCl₃) δ −141.37 (J=21.3), −138.10 (J=21.3).

(ii) Preparation of3-((3aS,4R,6S,6aR)-6-(2-azidoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine(1.b)

A mixture of2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethylmethanesulfonate (La) (0.641 g, 1 mmol) and sodium azide (0.070 mL, 2.00mmol) in DMF (7 mL) was heated to 60° C. for 15.5 h under nitrogenatmosphere. A white precipitate formed. Water was added (20 mL) and theproduct was extracted twice with TBME (100+40 mL). The organic phase wasdried over Na₂SO₄. The organic phase was filtrated and the solvent wasremoved under reduced pressure. The residue was purified by flashchromatography on a 2×8 cm silica column using heptane/EtOAc 1/1 aseluent, (TLC using heptane/EtOAc 1/1 (Rf product=0.5)). Collection ofthe relevant fractions and evaporation of the solvents gave the titlecompound (1.b) (514 mg, 87%) as a clear, thick oil.

¹H NMR (400 MHz, CDCl₃) δ 1.00 (s, 3H), 1.33-1.42 (m, 5H), 1.59 (s, 3H),1.73 (d, 2H), 2.17 (s, 1H), 2.68 (t, 2H), 2.96-3.17 (m, 3H), 3.19-3.33(m, 2H), 3.52-3.63 (m, 1H), 3.72 (ddd, 1H), 4.03 (td, 1H), 4.79 (dd,1H), 5.13 (td, 1H), 5.54 (dd, 1H), 6.43 (s, 1H), 6.96-7.23 (m, 3H).

(iii) Preparation of the Intermediate3-((3aS,4R,6S,6aR)-6-(2-aminoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine(1.c)

3-((3aS,4R,6S,6aR)-6-(2-azidoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine(1.b) (62.0 mg, 0.11 mmol) in EtOH (99.5%) (2 mL) was added to Pd/C (5%Pd, 50 wt % Pd/C, 22.46 mg, 5.28 μmol) and the mixture was hydrogenatedat atmospheric pressure for 2 h. The reaction mixture was filteredthrough HYFLO® and the plug was further rinsed with EtOH (99.5%). Thesolvent was removed under reduced pressure, the residue was re-dissolvedin DCM (2×2 mL) and the solvent was removed under reduced pressure. Theresidue was purified by flash chromatography on a 2×8 cm silica columnusing DCM/NH₃ (sat.) in MeOH 95/5 as eluent. Collection of the relevantfractions gave the title compound (1.c) (41 mg, 69%).

¹H NMR (400 MHz, CDCl₃) δ 0.98 (m, 3H), 1.28-1.46 (m, 7H), 1.54 (s, 3H),1.62-1.81 (m, 2H), 2.15 (s, 1H), 2.48-2.81 (m, 4H), 3.07 (tt, 3H),3.34-3.47 (m, 1H), 3.53 (ddd, 1H), 3.99 (td, 1H), 4.79 (dd, 1H), 5.12(td, 1H), 5.52 (dd, 1H), 7.02 (s, 1H), 7.09 (dt, 2H), 7.23 (s, 1H).

¹⁹F NMR (376 MHz, CDCl₃) δ −141.43 (J=21.3), −138.13 (J=21.3).

(iv) Preparation of the Intermediate(1S,2S,3S,5R)-3-(2-aminoethoxy)-5-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2-diol(1.d)

A pre-cooled, ice/water bath temperature, mixture of TFA (8 mL, 103.84mmol) and water (0.88 mL, 48.85 mmol) was added to a pre-cooled flaskwith3-((3aS,4R,6S,6aR)-6-(2-aminoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine(1.c) (340 mg, 0.61 mmol). The reaction mixture was stirred at 0-5° C.for 1 h. The solvent was removed under reduced pressure and the residuewas dissolved in DCM (100 mL) and washed with NaHCO₃ (sat. 10 mL). Brine(5 mL) was added to the aqueous phase and this was extracted with EtOAc(30 mL). The combined organic phases were dried over Na₂SO₄. Filtrationfollowed by evaporation of the solvents gave the crude product as anoff-white solid. The compound was purified by preparative HPLC on aXBridge C18 column (10 μm 250×50 ID mm) using a gradient of 35-75% ACNin H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minutes with a flow of 100mL/min. The compounds were detected by UV at 298 nm. The peak fractionswere evaporated to dryness under reduced pressure. The residue wasdissolved in DCM and filtered through a phase separator. Removal of thesolvent under reduced pressure gave the title compound (1.d) (213 mg,67.5%). LC-MS m/z 522.3 (M+H)⁺.

(v) Preparation of CompoundN-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,3-dihydroxycyclopentyl)oxy)ethyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide.(1.1)

2,5-dioxopyrrolidin-1-yl6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanoate(21.77 mg, 0.04 mmol) was added to a solution of(1S,2S,3S,5R)-3-(2-aminoethoxy)-5-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2-diol(20 mg, 0.04 mmol) in dry DMF (1.0 mL) and the mixture was placed underan nitrogen atmosphere and stirred at r.t. for 6 h. The solvent wasremoved under reduced pressure at 40° C. The compound was purified bypreparative HPLC on a Kromasil C18 column (10 μm 250×20 ID mm) using agradient of 20-60% ACN in H₂O/ACN/FA 95/5/0.2 buffer, over 20 minuteswith a flow of 19 mL/min. The compounds were detected by UV at 298 nm.The peak fractions were collected, concentrated, and freeze dried togive the title compound (1.1) (21.4 mg, 57.3%).

¹H NMR (600 MHz, DMSO): Two rotamers present (ratio 5:1), signals frommajor rotamer at δ 0.81 (t, 3H), 1.15-1.64 (m, 20H), 2.03 (ddd, 7H),2.12 (ddd, 1H), 2.57 (d, 1H), 2.59-2.67 (m, 1H), 2.77-2.89 (m, 2H), 2.93(dd, 1H), 2.96-3.01 (m, 4H), 3.05-3.12 (m, 1H), 3.15 (td, 1H), 3.18-3.25(m, 2H), 3.39-3.46 (m, 1H), 3.48 (tt, 1H), 3.7-3.76 (m, 1H), 3.92 (s,1H), 4.08-4.14 (m, 1H), 4.30 (dd, 1H), 4.54 (dd, 1H), 4.95 (q, 1H), 5.06(s, 1H), 5.13 (d, 1H), 6.35 (s, 1H), 6.42 (s, 1H), 7.07 (d, 1H), 7.31(ddt, 2H), 7.71 (dt, 2H), 7.82 (t, 1H), 9.36 (d, 1H). Selected signalsfrom minor rotamer at δ 0.98 (CH₃), 8.95 (ArNH).

HRMS Calcd for [C45H65F2N11O7S2]⁺: 974.4556; found: 974.4585 (M+H)⁺

Example 1.2: Biotinylated AdenosineN-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(1.2)

(i) Preparation ofN-(((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(1.e)

DMF (2 mL) was added to 2,5-dioxopyrrolidin-1-yl6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanoate(55.6 mg, 0.10 mmol) and9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(30 mg, 0.10 mmol) (See Austin, D. J. and Liu, F., Tetrahedr. Lett.,2001, 3153-3154) at r.t. and the reaction mixture was placed undernitrogen atmosphere and stirred for 1 h and 45 min to provide (1.e). Thesolvent was subsequently removed under reduced pressure. Used as crudeproduct without further purification. LC-MS m/z 759 (M+H)⁺, 757 (M−H)⁻.

(ii) Preparation of the Final CompoundN-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(1.2)

A mixture of TFA (1.8 ml, 23.36 mmol) and water (0.2 mL, 11.10 mmol) wasadded to the crudeN-(((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(1.e) (76 mg, 0.1 mmol) and the reaction mixture was stirred at 0° C.for 1 h 25 minutes. The solvents were removed under reduced pressure andthe residue was dissolved in DMSO. The compound was purified bypreparative HPLC on a XBridge C18 column (10 μm 250×19 ID mm) using agradient of 5-45% ACN in H₂O/ACN/NH₃ 95/5/0.2 buffer over 20 minuteswith a flow of 19 mL/min. The compounds were detected by UV at 259 nm.The peak fractions were concentrated and freeze dried to give the titlecompound (1.2) (50 mg, 69.6%) as a white fluffy solid.

¹H NMR (600 MHz, DMSO, 40° C.) δ 1.17-1.26 (m, 4H), 1.27-1.4 (m, 6H),1.43-1.54 (m, 7H), 1.62 (ddt, 1H), 1.99-2.06 (m, 4H), 2.12 (t, 2H), 2.58(d, 1H), 2.82 (dt, 1H), 2.96-3.05 (m, 4H), 3.05-3.14 (m, 1H), 3.36 (dt,1H), 3.44 (dt, 1H), 3.96 (dd, 1H), 4.04 (dd, 1H), 4.11-4.15 (m, 1H),4.29-4.33 (m, 1H), 4.67 (dd, 1H), 5.16 (d, 1H), 5.38 (d, 1H), 5.84 (d,1H), 6.29 (s, 1H), 6.33 (d, 1H), 7.25 (s, 2H), 7.64 (dt, 2H), 8.11 (t,1H), 8.16 (s, 1H), 8.31 (s, 1H). HRMS Calcd for [C32H50N10O7S]⁺:719.3657; found: 719.3667 (M+H)⁺

Example 2: Isolation and Identification of Anti-Ticagrelor/TAMAntibodies

This example illustrates strategies and techniques that may be used inthe production of antibodies to ticagrelor and its metabolites,compounds that have structural similarity to ATP and contains anadenosine-like core (Springthorpe et al 2007 Bioorg Med Chem Lett.17:6013-6018). The chemical structures of ticagrelor, ticagrelor activemetabolite (TAM) and ticagrelor inactive metabolite (TIM) are shown inFIG. 2 . As discussed above, the antibodies disclosed and generatedherein can bind and neutralise ticagrelor and TAM and may bind to TIM,but do not bind or significantly inhibit other structurally relatedcompounds such as adenosine. While binding activity to TIM is anoptional feature of the antibodies disclosed herein, antibodies thatexhibit binding activity to TIM are not expected to affect the dose ofthe antibody/antidote required, because TIM typically represents a smallor insignificant fraction of ticagrelor metabolites.

The common epitopes, i.e., unique R groups of ticagrelor and TAM(di-fluorophenyl-cyclopropyl and thiopropyl substituents) were targetedin order to confer antibody binding specificity and selectivity forthose compounds. The epitope of interest is encircled by the dashed linein FIG. 2 . The haptens described in Example 1 were used to direct theantibody epitope towards the di-fluorophenyl-cyclopropyl and thiopropylsubstituent groups. As described in Example 1, the linkers for thebiotinylated haptens (biotinylated ticagrelor and biotinylatedadenosine) were located on the glycol group. This strategy allowed forproduction of antibodies having binding specificity for the unmodifieddi-fluorophenyl-cyclopropyl and thiopropyl groups, for biotinylatedticagrelor/TAM, and also enabled for the screening and deselection ofantibody libraries that bind adenosine.

Using known techniques, a human scFv phage display library was used togenerate scFv antibodies, and specific scFvs were isolated from thelibrary in a series of repeated selection cycles on biotinylatedticagrelor with deselection on biotinylated adenosine, essentially asdescribed in Lloyd et al 2009 PEDS 22:159-168, incorporated herein byreference. A number of individual clones from the round 2 and round 3selection outputs were selected, scFvs were expressed in the bacterialperiplasm and screened for specificity in three parallel biochemicalassays. The assays screened for i) binding to biotinylated ticagrelor(Assay 1), ii) binding to biotinylated adenosine (Assay 2) and iii)binding to biotinylated ticagrelor in the presence of a 50-fold excessof unmodified ticagrelor (Assay 3) to confirm specificity for ticagrelorand not the biotinylated linker.

Assays 1, 2, and 3 were performed using the same general techniques andstrategy. Briefly, HTS of crude periplasmic scFv samples for binding tobiotinylated ticagrelor or to biotinylated adenosine was performed usingHTRF® assay technology. HTRF® (homogeneous time resolved fluorescence)is based upon the principle of TR-FRET (time resolved fluorescenceresonance energy transfer). Briefly TR-FRET utilizes the transfer ofenergy from a donor fluorophore (in this case Europium cryptate) to anacceptor fluorophore (in this case XL⁶⁶⁵). Provided the donor andacceptor fluorophores are in sufficiently close proximity (approx. <10nm), the excitation of the Europium cryptate donor (337 nm) results in atransfer of energy to the XL⁶⁶⁵ acceptor which in turn emits afluorescence signal at 665 nm. This technology can be used tosensitively measure biomolecular interactions by attaching the donor andacceptor fluorophores (either directly or indirectly) to each bindingpartner in the particular interaction. The HTS format for scFv bindingto biotinylated ticagrelor (Assay 1) is represented below and relies onthe presence of chemical tags on both the biotinylated ticagrelor andhis-tagged periplasmic scFv:

-   -   Europium crypate streptavidin:biotinylated        ticagrelor:scFv-His:anti-His-XL⁶⁶⁵

The assay was performed in a buffer comprising DPBS pH7.4 (Gibco14190-086), KF (VWR 103444T) (0.4M) and Tween 20 (Sigma P9416) (0.05%)in an assay volume of 10 ul using black shallow well 384 well assayplates (Corning/Costar 3676). The assay was set up by addition of 5 ulof biotinylated ticagrelor (60 nM to give 30 nM final concentration), 2ul of periplasmic scFv sample (20% final concentration) and 3 ul of asolution containing both Europium cryptate labelled streptavidin (CisBio610SAKLB) (4.2 nM to give 1.26 nM final concentration) and XL⁶⁶⁵labelled anti-His antibody (CisBio 61HISXLB) (40 nM to give 12 nM finalconcentration). Negative binding control wells were set up whichcontained all of the assay components above except that 2 ul assaybuffer was added in place of the periplasmic scFv. Assay plates wereincubated for 4 hrs at RT before being read on an Envision plate readerusing a standard HTRF read protocol in which samples are excited at 337nm and time resolved fluorescence emission is measured at both 620 nmand 665 nM.

Raw 665 nm and 620 nm counts were first converted into 665 nm/620 nmratio values and subsequently results were expressed as Delta F (%)values. Delta F was calculated according to the following equation:Delta F (%)={((sample 665/620 ratio)−(negative 665/620 ratio))/(negative665/620 ratio)}×100

(Negative ratio was taken from the negative binding control wells). scFvproviding Delta F values of greater than 100% were defined as hits inthis assay.

The same protocol as described above was used to perform HTS of crudeperiplasmic scFv samples for binding to biotinylated adenosine (finalassay concentration 30 nM). The format of Assay 2 can be depicted as setout below:

-   -   Europium crypate streptavidin:biotinylated        adenosine:scFv-His:anti-His-XL⁶⁶⁵

Assay 3 utilized the same protocol as described above to perform HTSthat identified crude periplasmic scFv samples showing reduced bindingto biotinylated ticagrelor in the presence of excess free unmodifiedticagrelor. This protocol was modified from Assay 1 in that Assay 3 wasperformed in the presence of a 50-fold molar excess of free, unmodifiedticagrelor (1500 nM).

A hit was defined as binding to biotinylated ticagrelor (Delta F>100% inAssay 1), no binding to biotinylated adenosine (Delta F<25% in Assay 2)and >50% reduced binding to biotinylated ticagrelor in the presence ofexcess free unmodified ticagrelor. An example correlation of data fromAssays 1 and 3 is shown in FIG. 3 . A number of scFvs showed limitedinhibition in the presence of excess free unmodified ticagrelor,implying they had binding interaction with some component of ticagrelorand the linker. A subset of scFvs were identified in which binding tobiotinylated ticagrelor was inhibited (>50%) in the presence of excessfree unmodified ticagrelor. scFvs were ranked based on the % inhibitionof binding observed in Assay 3 versus Assay 1 (50-80%, 80-90%, >90%)with scFv giving >90% inhibition (including TICA0072) prioritised forfurther characterisation. Sequence unique scFv hits were converted toFabs and expressed in CHO cells using standard techniques.

Fab Expression and Purification

Separate HC and LC expression plasmids were used for transienttransfection, based on the expression vectors described by Persic et al.1997. The vectors were modified to contain the EBV origin of replication(OriP). The Fab (HC) vector contained only constant region 1 (CH1) andHinge regions, CH2 and CH3 were removed. HC and LC DNA was added to 150mM NaCl and 25-kDa linear PEI (Polysciences Europe, Germany 23966),according to manufacturer's recommendations. The DNA-PEI complex wasthen added to Chinese Hamster Ovary wild type (CHO wt) cells derivedfrom a CHOK1 cell line (ECACC No: 85051005) adapted for suspensionculture (Daramola O, et al 2014). After 7 days the cells were harvestedby centrifugation and the supernatants filtered. The cell culturesupernatant containing the Fab protein was loaded directly onto achromatography column packed with 5 ml CaptureSelect IgG-CH1 (LifeTechnologies, Carlsbad, USA) at a flow rate of 5 mL/min using an AktaPurifier (GE Healthcare). The columns were equilibrated and washed withPhosphate Buffered Saline (PBS) pH 7.2 and eluted with 20 mM sodiumcitrate, 150 mM sodium chloride, pH 3.5 (CaptureSelect IgG-CH1)according to the resin manufacturer's instructions. Eluted Fab wasadjusted to pH 5.5 and filtered (0.22 μm Steriflip, Millipore EMD,Bethdesa, USA) prior to analysis. Protein concentration was determinedby absorbance at 280 nm using a DU520 UV/vis spectrophotometer (BeckmanCoulter, Brea, USA). Sample purity was determined using a TSKgelG3000SWxl column (Tosoh Bioscience, Tokyo, Japan) and a 1100 HPLC system(Agilent Technologies, Santa Clara, USA) running at 1.0 ml/min.

Example 3: Anti-Ticagrelor/TAM Fabs

A structural database (“DrugsDB”, Oprea T. I et al 2011 Mol. Inform.30(2-3), 100-111, incorporated herein by reference) containing marketeddrugs was interrogated in order to identify molecules that have somestructural similarity to ticagrelor. Once identified, these structurallysimilar molecules were used to test the binding specificity of Fabsbeyond adenosine and its phosphorylated forms (e.g., ADP and ATP). Thedatabase was interrogated for molecules having 2D fingerprintsimilarity, 3D shape, and electrostatic similarity to ticagrelor, basedon ticagrelor x-ray and NMR structures. From this in silico analysis apanel of 12 compounds were selected that included six potentialco-medications. The structures of these compounds are shown in FIG. 4 .

Specificity was investigated in a competition binding assay format inwhich the ability or otherwise of each test compound to competitivelyinhibit the interaction of biotinylated ticagrelor with the relevant Fabwas tested. A HTRF® competition assay format was used as set out belowin which the aim was to measure the competition of biotinylatedticagrelor binding to each His-Fab by a panel of test compounds:

Europium crypate anti-His antibody:test His-Fab:biotinylatedticagrelor:XL⁶⁶⁵ labelled streptavidin.

This basic assay format was used to assess the selectivity profile oflead Fabs from the end of both the lead isolation and lead optimizationphases and for the purposes of these studies Fabs were generated using aHis-Fab expression vector.

The assay was performed in a buffer comprising DPBS pH7.4 (Gibco14190-086), KF (VWR 103444T) (0.4M) and BSA (PAA K05-013) (0.1%) in anassay volume of 20 ul in black shallow well 384 well assay plates(Corning/Costar 3676). The assay set up involved the addition of 5 ul ofbiotinylated ticagrelor, 5 ul of a titration of each test selectivitycompound, 5 ul of the relevant His-Fab and 5 ul of a combined solutioncontaining both Europium cryptate labelled anti-His antibody (CisBio61HISKLB) (5.33 nM to give 1.33 nM final concentration) and XL⁶⁶⁵labelled streptavidin (CisBio 611SAXLB) (40 nM to give 10 nM finalconcentration). Total Binding control wells were set up which containedall of the assay components above except that 5 ul assay buffer wasadded in place of the test selectivity compound addition. NegativeBinding control wells were set up which contained all of the assaycomponents included in the Total Binding control wells except that 5 ulassay buffer was added in place of the His-Fab addition. Test compoundserial titrations were either ½ or ⅓ depending on the particularexperiment and top final assay compound concentrations were optimized ona compound specific basis. The concentration of biotinylated ticagrelorand His-Fab was optimized on a Fab specific basis in separateexperiments. For the four Fabs investigated at the end of the leadisolation phase (TICA0010, TICA0049, TICA0053 and TICA0072) the finalassay reagent concentrations used are set out in Table 1 below:

TABLE 1 Antibody ID [Antibody] (nM) [biotinylated ticagrelor] (nM)TICA0039 16.0 139.9 TICA0049 16.0 37.9 TICA0053 16.0 70.7 TICA0072 8.017.6

For the two Fabs investigated at the end of the lead optimization phase(TICA0162 and TICA0212) the final assay reagent concentrations used were5 nM biotinylated ticagrelor and 1 nM His Fab in both cases. Several ofthe test selectivity compounds used in these experiments were dissolvedin 100% DMSO and a vehicle related reduction in assay signal can beginto occur at concentrations above approximately 1% DMSO. In order toenable subsequent normalization of data to correct for any such vehiclerelated effects parallel titrations of DMSO alone were included in mostexperiments in which the final assay DMSO concentrations mirrored thoseof the test compound serial dilutions. At the end of the set upprocedure that assay was incubated for 3 hrs at RT before being read onan Envision plate reader using a standard read protocol.

For subsequent data analysis raw 665 nm and 620 nM counts were firstconverted into 665 nm/620 nm ratio values which were then used tocalculate % Delta F values according to the equation set out in Assay 1.The negative ratio value used in the calculation of % Delta F wasderived from the Negative Binding control wells. % Delta F values werethen used to calculate % Specific Binding values according to theequation below:Specific Binding (%)={(Sample Delta F−Negative Binding Delta F)/(TotalBinding Delta F−Negative Binding Delta F)}×100

For standard calculations of % Specific Binding Total Binding Delta Fwas taken from Total Binding control wells which contained all of theaforementioned components of the assay but without inclusion of anycompeting test compound.

In those experiments where DMSO normalization was applied DMSOnormalized % Specific Binding was calculated essentially according tothe equation above except that in this case Total Binding Delta F wastaken from wells containing all of the components in the Total Bindingcontrol wells as well as DMSO at a concentration equivalent to that inthe relevant sample well.

The results from initial experiments of this type are summarized inTable 2. In three of the four initial Fabs that were investigated(TICA0010, TICA0049 and TICA0053) the compound Cangrelor showedcompetitive inhibition of Fab binding to biotinylated ticagrelor. In thecase of TICA0049 both Pantoprazole and Linezolid exhibited partialcompetitive inhibition. As shown in Table 2, TICA0072 Fab exhibited noinhibition with eleven of the twelve compounds, with pantoprazoleshowing weak, partial inhibition.

Inhibition with unmodified ticagrelor and TAM was observed for all fourFabs tested in this first series of experiments with IC₅₀ values fallingin a range of 0.1 μM to 0.5 μM. Competitive inhibition was also detectedwith TIM for two of the four Fabs (TICA0049 and TICA0072) however, withIC₅₀ values of >50 μM, suggesting a greatly reduced affinity for TIMrelative to unmodified ticagrelor and TAM. Based on the results in Table2 TICA0072 was identified as having the most favorable selectivityprofile. TICA0049 was identified as a potential back up based on thecriteria that this Fab showed the least binding to Cangrelor within theremaining three Fabs.

TABLE 2 Relative IC₅₀ values for each of 12 tested compounds (FIG. 4)and unmodified ticagrelor, TAM, and TIM for inhibition of biotinylatedticagrelor binding to each test Fab. IC50 (uM) for Various Compounds forlead his-Fabs Compound TICAOOlO TICA0049 TICA0053 TICA0072 FenofibrateNI NI NI NI Nilvadipine NI NI NI NI Cilostazol NI NI NI NI BucladesineNI NI NI NI Regadenoson NI NI NI NI Cyclothiazide NI NI NI NI CyfluthrinNI NI NI NI Lovastatin NI NI NI NI Linezolid NI 467.1 NI NI SimvastatinNI NI NI NI Cangrelor 102.4   207.9 17.5   NI Pantoprazole NI 263.7 NI498.0 Ticagrelor 0.122 0.257 0.109 0.113 TAM 0.124 0.412 0.134 0.299 TIMNI 53.4 NI 54.4 NI is no inhibition.

In a second series of experiments further selectivity data was generatedfor TICA0049 and TICA0072 Fabs in which a subset of four of the twelvecompounds listed in FIG. 4 were retested along with ticagrelor, TAM andTIM and several adenosine related compounds. Here, in a refinement tothe earlier investigations in Table 2, experiments were designed inorder to enable normalisation of percent specific binding values tocorrect for any non-specific vehicle related effects due to DMSO.Example plotted data from this second series of experiments is shown inFIG. 5 along with tabulated IC₅₀ values in Table 3.

TABLE 3 Example IC₅₀ results for a subset of four of the twelvecompounds listed in FIG. 4, ticagrelor, TAM, TIM and several adenosinefamily compounds in competition binding selectivity studies (DMSOnormalised) Compound TICA0049 TICA0072 Adenosine NI NI ADP NI NI 2MeSADP NI NI ATP NI NI 2MeSATP NI NI Bucladesine 864.9 NI Linezolid 902.0NI Cangrelor 188.4 NI Pantoprazole 243.5 1546.0 Ticagrelor 0.368 0.356TAM 0.366 0.483 TIM 74.8 119.5

As with the earlier experiments TICA0072 showed the most favourableselectivity profile with only pantoprazole showing weak, partialinhibition (IC₅₀>1500 μM) from within the four compounds tested. In thecase of TICA0049 significant inhibition was observed with Cangrelor andPantoprazole with weak inhibition observed for Linezolid andBucladesine. It should be noted that subtle differences in absolute IC₅₀values between the first and second series of experiments for certaintest compounds do not fundamentally change the overall conclusions. Suchdifferences may stem from the fact that DMSO normalisation wasincorporated into the second series of experiments in combination withthe fact that in several cases we were attempting to measure very weakinhibition.

For both TICA0049 and TICA0072 measured IC₅₀ values for ticagrelor andTAM were in the range 0.3 μM to 0.5 μM with IC₅₀ values for TIM atgreater than 2 log values higher at 74.8 μM and 119.5 μM, respectively.A trace of inhibition was observed with adenosine, ADP, ATP and themethyl-thio derivatives of the latter two compounds for both TICA0049and TICA0072 Fabs however this was only detected at the highest compoundconcentrations tested and was not considered significant.

It is concluded that TICA0072 was the only Fab considered specific forticagrelor and TAM.

Example 4: Affinity Measurement of Anti-Ticagrelor/TAM Fabs

The affinity of the anti-ticagrelor Fabs generated above was determinedusing Biolayer Interferometry on the Octet Red384. For the affinitymeasurement anti-ticagrelor Fab antibodies were diluted to aconcentration of 2× the final assay concentration in assay buffer (PBS,Tween20 0.05%, BSA 0.02%) e.g. 200 nM. A 10-point 2-fold serial dilutionof ticagrelor was prepared in a Greiner polypropylene 96-well plate.Equal volumes, (e.g. 70 μL plus 70 μL) of diluted antibody and freeticagrelor were then transferred to a second Greiner polypropyleneplate. The samples were mixed by pipetting, covered with a plate sealand allowed to equilibrate for 3-5 days at room temperature. Followingequilibration 60 μL of the antibody/ticagrelor titrations wastransferred, in duplicate, to a 384 well black tilted bottompolypropylene plate. Biotinylated ticagrelor was diluted to 250 nM inassay buffer and added to alternate wells of the first 2 columns of the384 well assay plate, the remaining wells contained assay buffer only.Streptavidin biosensors were pre-soaked in assay buffer for at least 10minutes. Sample plates and biosensors were then loaded onto the stage ofthe OctetRed384.

All assays were performed at room temperature. Following baselineequilibration for 60 sec in assay buffer, biotinylated ticagrelor wasloaded on the streptavidin biosensors for 300 sec, followed by assaybuffer for 600 sec to establish a new baseline. The antibody/ticagrelormixtures were then allowed to associate with the biotinylated ticagrelorsensor surface for 30-600 sec, depending on the concentration ofantibody used. The resulting association phase data were analyzed usingthe OctetRed Data Analysis software. The signals were aligned tobaseline and the reference sensor signal (no antibody control) wassubtracted for each sample, then the data were exported for analysisusing the KinExA n-Curve Analysis software. Using the Constant Partneranalysis the equilibrium KD for the anti-ticagrelor Fab antibodies wasdetermined. The data showed that Fabs TICA0072 and TICA0049 hadaffinities for ticagrelor of 7.4 nM and 11.6 nM respectively (Table 4).

TABLE 4 Equilibrium affinity analysis for anti-ticagrelor Fabs 95%Confidence Antibody ID Hapten Equilibrium KD Interval TICA0072Ticagrelor 7.43 nM 1.75-21.46 nM  TICA0049 Ticagrelor 11.6 nM 1.7-66.5nM

Example 5: Optimization of Anti-Ticagrelor/TAM Antibody TICA0072

The antibody TICA0072 was optimized using affinity-based phageselections. Large scFv libraries derived from the lead scFv sequencewere created by oligonucleotide-directed mutagenesis of the variableheavy (VH) complementarity determining regions (CDR) 1, 2 or 3 orvariable light (VL) chain CDRs 1, 2 or 3 using standard molecularbiology techniques as described (Clackson and Lowman 2004 PracticalApproach Series 266). The libraries were subjected to affinity-basedphage display selections in order to select variants with a higheraffinity to ticagrelor and TAM. In brief, the scFv-phage particles wereincubated in solution with reducing concentrations of biotinylatedticagrelor (a typical example would be 20 nM to 20 pM over four roundsof selection), essentially as described previously (Thompson et al 1996J Mol Biol. 256 (1):77-88). Crude scFv-containing periplasmic extractswere prepared for a representative number of individual scFv from theCDR-targeted selection outputs and screened in a HTRF© epitopecompetition assay format designed to screen for improvements in affinityrelative to TICA072.

Briefly, in order to screen for scFv and Fab variants of improvedaffinity a HTRF® epitope competition assay was implemented based oncompetition, by test scFv variants, of the interaction between theparent TICA0072 IgG and biotinylated ticagrelor. This assay was usedboth as a primary single point HTS to screen crude periplasmic extractscFv samples as well as a multi-point secondary profiling assay in orderto measure improvements in IC₅₀ values for both purified scFv and Fabvariants versus parent TICA0072. Although epitope competition assays,such as the one described here, are not generally used to determineabsolute affinity values such assays can be used as the basis of anaffinity based HTS. Furthermore, fold improvements in IC₅₀ for purifiedscFv/Fab variants (relative to the parent scFv/Fab) can provide a goodindicator of overall fold gains in affinity and can represent aneffective way to affinity rank scFv/Fab variants in lead optimizationcampaigns. The format for the TICA0072 parent IgG based epitopecompetition assay described here is set out below:

Europium labelled streptavidin:biotinylated ticagrelor:TICA0072IgG:XL⁶⁶⁵ labelled anti-human-Fc antibody

The assay was performed in a buffer comprising DPBS pH7.4 (Gibco14190-086), KF (VWR 103444T) (0.4M) and Tween 20 (Sigma P9416) (0.05%)in black shallow well 384 well assay plates (Corning/Costar 3676). Forsingle point testing of crude periplasmic scFv variants an assay volumeof 10 ul was used however a 20 ul assay volume was used when testingpurified scFv and Fab in multipoint secondary IC₅₀ profiling assays.

For single point HTS the assay was set up by adding 3 ul of TICA0072 IgG(53.3 nM to give 16 nM final concentration), 2 ul of crude periplasmicextract scFv sample, 2.5 ul of biotinylated ticagrelor (8 nM to give 2nM final concentration) and 2.5 ul of a combined solution containingEuropium labelled streptavidin (CisBio 610SAKLB) (3 nM for 0.75 nM finalassay concentration) and XL⁶⁶⁵ labelled anti-human-Fc antibody (CisBio61HFCXLB) (30 nM to give 7.5 nM final assay concentration). ParentTICA0072 crude periplasmic scFv was used as benchmark and the HTS wasconfigured to identify variants giving improved inhibition relative toparent. Total Binding Control Wells contained all assay componentsexcept that 2 ul assay buffer was added in place of the scFv sample.Negative Binding control wells contained all of the components of theTotal Binding control wells except that 3 ul of assay buffer was addedin place of the TICA0072 IgG.

For multipoint IC₅₀ testing of purified scFv/Fab variants the assay wasset up by adding 5 ul of TICA0072 IgG (53.3 nM to give 16 nM finalconcentration), 5 ul of a ⅓ titration of purified test scFv or Fabvariant, 5 ul of biotinylated ticagrelor (8 nM to give 2 nM finalconcentration (scFv profiling), 4 nM to give 1 nM final assayconcentration (Fab profiling)) and 5 ul of a combined solutioncontaining Europium labelled streptavidin (CisBio 610SAKLB) (3 nM for0.75 nM final assay concentration) and XL⁶⁶⁵ labelled anti-human-Fcantibody (CisBio 61HFCXLB) (30 nM to give 7.5 nM final assayconcentration). Purified parent TICA0072 (scFv or Fab) was used as abenchmark in all experiments such that improvements in IC₅₀ measuredwith optimized variants (scFv or Fab) could be expressed as foldimprovements over parent TICA0072. Total Binding Control Wells containedall assay components except that 5 ul assay buffer was added in place ofthe purified scFv or Fab sample. Negative Binding control wellscontained all of the components of the Total Binding control wellsexcept that 5 ul of assay buffer was added in place of the TICA0072 IgG.

In both single point HTS and multipoint IC₅₀ profiling versions of theassay, plates were incubated for 3 hrs at RT before being read on anEnvision plate reader using a standard HTRF read protocol in whichsamples are excited at 337 nm and time resolved fluorescence emission ismeasured at both 620 nm and 665 nM.

Raw 665 nm and 620 nm counts were used to calculate Delta F (%) and %Specific Binding according to the equations described earlier in Assays1 and 4, respectively. For multipoint secondary profiling experimentsIC₅₀ values were determined using Graphpad Prism software usingsigmoidal dose response (variable slope) curve fitting (4 parameterlogistic equation).

Hits identified in the screen, i.e. scFv variants which showed asignificantly improved inhibitory effect when compared to parentTICA0072 scFv, were subjected to DNA sequencing, and unique variantsfrom variable heavy CDR1, CDR2 or CDR3 and variable light library CDR1,CDR2 or CDR3 outputs were then produced as purified scFv and retested inthe same assay to determine concentration response IC₅₀ curves. The scFvvariants showing the most improved IC₅₀ values were then produced asFabs and tested in a second generation epitope competition assay,described below.

Second Generation Epitope Competition Assay for Screening Ranking ofHighest Affinity Fab

In order to discriminate effectively between very high affinity purifiedFab at the end of the lead optimization campaign a further HTRF© epitopecompetition assay was implemented however in this case the assay wasbased upon competitive inhibition of an intermediate affinity optimizedTICA0072 lineage IgG (TICA0159) binding to biotinylated ticagrelor asopposed the parent TICA0072 IgG. This assay was used only in multipointIC₅₀ profiling format (as opposed to HTS format) and was performedessentially identically to the method given in Assay 5 (above) formultipoint IC₅₀ profiling of purified Fab variants in the parentTICA0072 IgG based epitope competition assay. The only difference beingthat TICA0159 IgG was used in place of TICA0072 IgG at the appropriatepoint in the assay set up but at the same 16 nM final assayconcentration. In all other respects this assay was performed exactly asdescribed above in the multipoint secondary profiling version forpurified Fab.

The second generation assay replaced TICA0072 with a partially optimizedantibody TICA0159 to enable more effective discrimination and ranking ofthe highest affinity Fabs than was possible in the TICA0072 basedepitope competition assay.

The most improved VH was identified as CDR3 variant TICA0162. The mostimproved VL was identified as CDR3 variant TICA0152. To generate furtheraffinity improvement the different CDRs from improved antibodies werecombined into new Fabs using standard molecular biology techniques. Fromthis recombination work the combination of TICA0162 and TICA0152generated a new Fab TICA0212 with a further improved epitope competitionprofile. Plotted competition curves for TICA0072, TICA0152, TICA0162 andTICA0212 Fab in the second generation epitope competition assay areshown in FIG. 6 with measured IC₅₀ values in Table 5. TICA0212 showsapproximately 2 log improvement in IC₅₀ relative to the parent TICA0072Fab.

TABLE 5 IC₅₀ data for listed optimized anti-ticagrelor Fabs in thesecond generation epitope competition assay. Fab IC₅₀ (nM) Fold ImprovedTICA0072 1714.0 0 TICA0152 73.5 23.3 TICA0162 16.3 105.2 TICA0212 12.0142.8

Example 6: Affinity Measurement of Optimized Anti-Ticagrelor/TAM Fabs

The affinity of anti-ticagrelor/TAM Fabs generated in Example 5 wasdetermined using the KinExA3200. For the KinExA affinity measurement,beads (600 mg Azlactone beads) were first prepared by reacting themovernight with 1 mg streptavidin in 50 mM NaHCO₃. Following blockingwith 2 changes of Tris buffer (1M Tris pH 8.7, 10 mg/mL BSA) thestreptavidin coated beads were resuspended in a total volume of 8 mL.Beads (1.33 mL, equivalent to 100 mg original dry Azlactone beads) werewashed completely with PBS, then allowed to bind to approximately 2.5 μgbiotin-ticagrelor in 1 mL PBS for 10 minutes with occasional agitation.The resulting biotin-ticagrelor coated beads were washed with PBS, thenresuspended in 50 mL PBS containing 0.1% BSA and 0.02% NaN₃ and storedat room temperature until transfer to the KinExA bead vial.

Antibody/ticagrelor sample preparation was carried out essentially asdescribed in the previous method. Anti-ticagrelor Fab antibodies werediluted to a concentration of 2 times the final assay concentration inassay buffer (PBS, Tween20 0.05%, BSA 0.02%, 0.02% NaN₃) e.g. 200 nM. A10-point 2-fold serial dilution of ticagrelor was prepared in Falcon 50mL polypropylene tubes. Equal volumes, e.g. 5 mL plus 5 mL, of dilutedantibody and free ticagrelor were then transferred to a second Falconpolypropylene tube. The samples were mixed by pipetting and allowed toequilibrate for 3-5 days at room temperature. Following equilibrationthe sample tubes were transferred to the KinExA 3200 for analysis.

All assays were performed at room temperature. The antibody/ticagrelormixtures were sampled and allowed to mix with the bio-ticagrelor beadswhile the unbound free ticagrelor was washed away. Bound antibody wasthen detected using DyLight649 labelled mouse anti-human Heavy and Lightchain antibody. Sample volume (300-1300 μL) and injection time (90-120s) varied with concentration, and 2-3 reads were varied out per sample.The data were analysed using the KinExA n-Curve Analysis software. Usingthe Constant Partner analysis the equilibrium KD for the anti-ticagrelorFab antibodies was determined.

As in the prior Example, Fabs were allowed to equilibrate at roomtemperature in the presence of either unmodified ticagrelor or TAM atconcentrations from 20-fold excess. Biotinylated ticagrelor was loadedon to the surface of streptavidin coated beads. Following equilibration,the remaining free antibody was allowed to bind to the biotinylatedticagrelor. Bound antibody was then detected using a DyLight649 labelledmouse anti-human Heavy and Light chain detection antibody. Titrations offree ticagrelor or TAM were prepared with at least three different fixedconcentrations of antibody to generate separate titration profiles withat least a 10-fold shift in apparent KD. The data were analysed usingthe KinExA Pro n-Curve analysis software to determine equilibrium KD forfree ticagrelor or TAM. Fab TICA0212 had an affinity for unmodifiedticagrelor and TAM of around 20 pM (Table 6). From the equilibrium dataTICA0212 demonstrates equivalent high affinity (˜20 pM) binding toticagrelor and TAM.

TABLE 6 Equilibrium affinity analysis for anti-Ticagrelor/TAM FabsAntibody Equilibrium ID CDR3 Seqs. (SEQ ID NO) Hapten KD 95% C. I.TICA0072 VH GSHLY⁹⁹ DFW^(100b) SASHPPNDALAI (35) Ticagrelor 7.4 nM1.8-21.5 nM VL GTW⁹¹ D⁹²I⁹³S⁹⁴LSAGL (40) TICA0152VH GSHLYDFWSASHPPNDALAI (65) Ticagrelor 43.17 pM 2.8-119.2 pMVL GTWLYDRAVGL (70) TICA0162 VH GSFDYYFWSASHPPNDALAI (55) Ticagrelor162.48 pM 125.4-206.3 pM VL GTWDISLSAGL (60) TICA0212VH GSFDYYFWSASHPPNDALAI (75) Ticagrelor 19.6 pM 13.0-28.7 pM (MEDI2452)VL GTWLYDRAVGL (80) TAM 19.7 pM 4.9-44.7 pM TIM ~20 Nm Changes insequence residues from parental TICA0072 are bolded, and kabat numbersare identified for certain residues in TICA0072.

Example 7: Specificity of Anti-Ticagrelor/TAM Fab TICA0162 and TICA212

The specificity of the Fabs, TICA0162 and TICA0212, was tested as inexample 3 and normalised against DMSO. A summary table of all availableselectivity data for TICA0162 and TICA0212 is included in Table 7. Inaddition example plotted data from an experiment in which five of thetwelve compounds listed in FIG. 4 were tested alongside ticagrelor, TAM,TIM and several adenosine family compounds is shown in FIG. 7 .

TABLE 7 Relative IC₅₀ values of the twelve structurally relatedcompounds in addition to ticagrelor, TAM, TIM and adenosine familycompounds for inhibition of biotinylated ticagrelor binding to TICA0162and TICA0212 Fab. IC50 (uM) for Various Compounds for lead his-FabsCompound TICA0162 TICA0212 Fenofibrate NI NI Nilvadipine NI NICilostazol NI (n = 2) NI (n = 2) Bucladesine NI NI Regadenoson NI (n =2) NI (n = 2) Cyclothiazide >1000 (n = 2)   NI (n = 2) Cyfluthrin NI NILovastatin NI NI Linezolid NI NI Simvastatin NI (n = 2) NI (n = 2)Cangrelor NI NI Pantoprazole >1000 (n = 2)   NI (n = 2) Adenosine NI NIADP NI NI 2MeS-APD NI NI ATP NI NI 2MeS-ATP NI NI Ticagrelor 0.023 (n =2)   0.035 (n = 2)   TAM 0.032 (n = 2)   0.031 (n = 2)   TIM 19.8 (n =2)  28.8 (n = 2)  NI is no inhibition.

As illustrated by the data TICA0212 (MEDI2452) has essentiallyequivalent binding specificity to ticagrelor and TAM, and weaker bindingto TIM. In addition MED12452/TICA0212 did not show significant bindingto any other structurally related drugs or adenosine related compounds.

Example 8: Protein Crystallography

TICA0072 with a C-terminal his-tag was concentrated in PBS to 9 mg/ml.Complex formation was achieved by the addition of 1 mM ticagrelordissolved in DMSO. The complex was incubated in room temperature for 2hours before crystallization trials were set up using the vapordiffusion method with sitting drops. Broad screening was performed using3 commercial screens and several hits were obtained. Best diffractingcrystals were obtained from a grid optimization of a Morpheus®(Molecular Dimensions, UK) hit. The crystals used for structuredetermination was grown at 20° C. by mixing equal volumes ofTICA0072-ticagrelor complex and a reservoir solution of 12.8% PEG 3350,12.8% PEG 1000, 12.8% MPD, 1.7% 1,6-Hexanediol, 1.7% 1-Butanol, 1.7%1,2-Propanediol, 1.7% 2-Propanol, 1.7% 1,4 butanediol, 1.7%1,3-Propanediol, 25 mM Imidazole, 25 mM Sodium Cacodylate. 25 mM MES and25 mM Bis-Tris pH 6.5. Crystals were flash-frozen in liquid nitrogenwithout the addition of any cryo protectant.

TICA0212/MED12452 in PBS at a concentration of around 15 mg/ml was mixedwith Ticagrelor to a concentration of 1 mM and incubated 2 hours at roomtemperature before broad screening was performed. No spontaneous hitswere obtained. Seeds from TICA0072-Ticagrelor crystals were prepared bycrushing several crystals in 30 ul well solution and used in MMS(microseed matrix screening) into broad commercial screens. Several hitswere obtained but the crystals used for structure determination wasgrown at 20° C. from a condition of 20% glycerol, 20% PEG 4000, 10%2-propanol, 0.1 M NaCl and 0.5 M NaAcetate pH 4.6. The drops were set upwith 0.2 ul protein, 0.18 ul well solution and 0.02 ul seed stock.Crystals were flash-frozen in liquid nitrogen without the addition ofany cryo protectant.

Data were collected at beam line ID23-1 at the European SynchrotronRadiation Facility, Grenoble, France. The data were processed, scaledand further reduced using the AutoProc workflow [Vonrhein, C., et al.,Acta Cryst. 2011; D67: 293-302], for statistics see Table 8. ForTICA0072 initial phasing was done by molecular replacement using a highresolution Fab structure (PDB id code lagk, [Faber C., et al.,Immunotechnology. 1998; 3:253-70]) as a starting model. ForTICA0212/MED12452, the structure for TICA0072 was used as a startingmodel. Model rebuilding was performed using Coot [Bricogne G., et al.,(2011). BUSTER version 2.11.4. Cambridge, United Kingdom: Global PhasingLtd] and refinement was performed using autobuster [Emsley, P., et al.,Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, D60, 2126-2132].For statistics for the final models see Table 8.

TABLE 8 Data collection and refinement statistics from Fab ticagrelorco-crystal structures. Values in parenthesis refer to highest-resolutionshell. TICA0212 Description TICA0072 (MEDI2452) PDB accession code Datacollection statistics Radiation source ESRF/ID23-1 ESRF/ID23-1 Radiationdetector Pilatus Pilatus Space group P21 P21212 Cell dimensions a =41.2, b = 72.6, a = 69, b = 173, c = 67.8 β = 98.9 c = 42 Resolution (Å)49-1.7 (1.87-1.7) 41-2.16 (2.27-2.16) Observed reflections 143002 232480Unique reflections 41888 36570 Completeness (%) 97.0 (95.3) 99.4 (94.7)Mean I/σ_(I) 13.6 (1.7) 10.7 (1.2) R_(sym) %^(b) 4.8 (74.4) 9.7 (27.7)Refinement statistics Resolution (Å) 49-1.7 41-2.16 No. protein + ligandatoms 3308 3371 No. solvent atoms 142 87 R (%), Rfree (%) 19.8, 23.721.7, 25.7 Wilson B (Å²), 45.6, 48.5 42.1, 44.6 Refined <B> (Å²) Rmsdideal bond lengths 0.008 0.010 (Å) Bond angles (°) 1.10 1.25

The structure of TICA0072 in complex with ticagrelor was determined to1.7 Å resolution (FIG. 10 ). The CDRs form a highly concave surface andticagrelor is inserted deep into the interface between VH and VLdomains. This type of binding is commonly observed for small haptens.All CDRs except VL CDR2 are contributing directly to ticagrelor bindingand a large portion of VH CDR3 is disordered. Ticagrelor'sdifluorophenyl group is located in a cavity lined with hydrophobicresidues including vernier residues VH Trp47, VL Phe98 and VH CDR3residue Leu100L. A key residue in the interaction with ticagrelor is VLTrp91, which is involved both in a pi-stacking against theadenosine-like core and a hydrogen bond to one of the ribose hydroxylgroups at the cyclopentyl moiety. Additional interactions to theadenosine-like core are provided by VH CDR1 His35 and VH CDR3 Tyr99. Thethiopropyl substituent stacks against the main chain of the VH CDR2loop. The hydroxyethyl substituent on the cyclopentyl moiety isprotruding into the solvent and does not make any interactions with theFab.

In the structure of the affinity improved Fab, TICA0212/MED12452, theticagrelor binding is similar to that of TICA0072 with all theinteractions mentioned above retained but with some importantdifferences (FIG. 10B). The combination of the VL CDR3 mutationsAsp92Leu and Ser94Asp breaks a hydrogen bond within the VL CDR3 loop tocreate a more “relaxed” structure. The new conformation is correlatedwith a 15° tilt of the pyrimidine ring about the attachment of thecyclopropyl-difluorophenyl substituent. The new position of thepyrimidine ring brings it approximately 0.2 Å closer to VH CDR3 Tyr99 inTICA0212/MED12452 compared to Fab 72. Moreover, VL Ile93Tyr introduces ahydrogen bond donor which makes interactions with the VH CDR3 loop thusdefining the binding site further.

The crystal structure for TICA0212/MED12452 shows ticagrelor bound in adeep crevice between the V_(H) and V_(L) interface. The design strategyof labelling ticagrelor with biotin via a tri-amide linker to thehydroxyethyl group is substantiated as the crystal structuredemonstrates the hydroxyethyl group is not involved in any interactionwith TICA0212/MED12452. This is further supported by the fact that theFab also binds TAM with identical affinity, which lacks the hydroxyethylgroup. TICA0212/MED12452 shows weak binding to TIM which lacks thecyclopropyl-difluorophenyl group. In the TICA0212/MED12452-ticagrelorcomplex the cyclopropyl-difluorophenyl group is buried at the bottom ofthe hydrophobic pocket and must play a key structural role in aligningthe ticagrelor adenosine-like core with the V_(L) CDR3 residue Trp L91.As the chemical starting point for ticagrelor was ATP and it retains anadenosine-like core, a critical attribute for antidote specificity wasto demonstrate no binding of adenosine. The lead isolation strategyinvolved both high throughput and detailed specificity analysis and nobinding of adenosine was detected either by competitive or directbinding analyses. From the structural analysis the purine ring andribose group of adenosine might be expected to mimic the interaction ofticagrelor's adenosine-like core. However, the lack of binding may beexplained by the absence of two hydrophobic R groups(cyclopropyl-difluorophenyl and thiopropyl) which significantly reduceboth the relative shape complementarity and the hydrophobicity of thebinding interaction.

An analysis of the structures of the parental TICA0072 andTICA0212/MEDI2452 shows the significance of some of the changesintroduced during the affinity maturation. The mutations in V_(L) CDR3appear to have particularly high impact, resulting in a different loopconformation and an additional hydrogen bond in TICA0212/MEDI2452 todefine the binding cavity. In contrast, contributions from mutations inthe VH CDR3 are structurally less obvious. These observations from thestructure are partly confirmed by data for modified antibodiescontaining modifications in V_(L) CDR3 (TICA0152) or V_(H)CDR3(TICA0162) only, which resulted in 200-fold and 50-fold improvement overTICA0072, respectively. However, although the V_(L) CDR3 changes appearto have a greater effect, both sets of mutations add significantimprovement. It should be noted that the crystal structure is a staticpicture of the complex and is not able to capture any of the protein andligand dynamics involved in binding.

Example 9: TICA0212/MEDI2452 Concentration-Dependently Restored PlateletAggregation in the Presence of Ticagrelor or TAM, In Vitro

The extent and potency of TICA0212/MEDI2452 to reverse ticagrelor or TAMmediated inhibition of ADP-induced platelet aggregation was determinedin human platelet rich plasma (PRP) using light transmissionaggregometry.

For the in vitro human PRP assay, blood was collected from fastedhealthy volunteers by vein puncture of vena cephalica. The first 2 mL ofblood was discarded prior to collecting aliquots into tubes containing0.109 M sodium citrate, 1+9 (citrate+blood), to final concentration 10.9mM. The anticoagulated human blood was centrifuged at 240×g for 15 min.PRP was carefully removed and transferred to a clean vial. Platelet poorplasma (PPP) was prepared by centrifugation of the PRP at 2000×g for 15min. Light transmission aggregometry (LTA) was evaluated in PRP by thePlatelet Aggregation Profiler (PAP-8E, Bio/Data Corporation, Pa., USA.).Zero % aggregation was defined as the light transmission of PRP and 100%aggregation was defined as the light transmission of PPP.

PRP was preincubated with 1 μM ticagrelor or TAM for 1 hour beforeco-incubating with different concentrations of TICA0212 or an isotypecontrol Fab for 30 minutes. Platelet aggregation was initiated byaddition of 20 μM ADP and was continuously recorded for 6 min. Data forfinal aggregation (FA) extent at 6 min was analysed.

The concentrations of TICA0212/MEDI2452 that gave half-maximum reversal(IC₅₀) were calculated. TICA0212/MEDI2452 produced aconcentration-dependent reversal of 1 μM ticagrelor and 1 μM TAMmediated inhibition of 20 μM ADP-induced platelet aggregation withcalculated mean (n=5) IC₅₀ values of 0.64 and 0.78 μM, respectively(FIG. 8 ). When evaluated at 1:1 conditions (1 μM TICA0212/MED12452: 1μM ticagrelor or TAM) the mean extent of reversal was 78% and 62%,respectively. Isotype control Fab caused no significant reversal ofticagrelor and TAM ADP-induced platelet aggregation. For example, after30 min incubation isotype control Fab resulted in −3% and 2% reversal ofticagrelor and TAM, respectively.

From this, the data shows that TICA0212/MEDI2452 can reverse bothticagrelor and TAM mediated inhibition of ADP-induced plateletaggregation in vitro in a concentration dependent manner. Maximal andnearly complete reversal effect was achieved when evaluated in a 1:1experiment setting, which would be predicted when TICA0212/MEDI2452binds to ticagrelor or TAM in a 1:1 stoichiometry.

Example 10: TICA0212/MEDI2452 Effectively and Rapidly Restores PlateletAggregation in the Presence of Ticagrelor or TAM, In Vitro

The time of onset of TICA0212/MED12452 to reverse ticagrelor or TAM wasdetermined in human platelet rich plasma (PRP) using light transmissionaggregometry as in example 8. PRP was preincubated with 1 μM ticagreloror TAM for 1 hour before adding 1 μM TICA0212/MEDI2452 and co-incubatingfor 5, 10, 15, 30 and 60 min, or adding an isotype control Fab for 30min. Platelet aggregation was initiated by addition of 20 μM ADP and wascontinuously recorded for 6 min. Data for final aggregation (FA) extentat 6 min was analyzed.

TICA0212/MED12452 produced similar extent of reversal of ticagrelormediated inhibition regardless of co-incubation time as mean (n=3)extent of reversal was 85%, 69%, 74%, 80% and 81% after 5, 10, 15, 30and 60 minutes, respectively. Similarly the mean (n=3) extent of TAMreversal induced by TICA0212/MEDI2452 was 53%, 56%, 58%, 69%, 74% after5, 10, 15, 30 and 60 minutes, respectively. TICA0212/MEDI2452 rapidlyand effectively reversed ticagrelor and mediated inhibition ofADP-induced platelet aggregation. No reversal, mean (n=3) −2%, waspresent after 30 min co-incubation with the isotype control Fab. Fromthis data, TICA0212/MEDI2452 was shown to rapidly and effectivelyreverse both ticagrelor and TAM mediated inhibition of ADP-inducedaggregation when evaluated in a 1:1 experiment setting.

Example 11: TICA0212/MEDI2452 Effectively and Rapidly Restored PlateletAggregation after Dosing to Ticagrelor Treated Mice, In Vivo

The speed of onset and the extent of TICA0212/MEDI2452 mediated reversalof ticagrelor mediated inhibition of ADP-induced platelet aggregation inwhole blood (impedance aggregometry) was determined ex vivo afterintravenous (i.v.) dosing of ticagrelor in mice. Mice were giventicagrelor i.v. as a bolus of 1200 μg/kg, over 5 minutes, followed by a15 min continuous infusion of 30 μg/kg/min. After termination of theticagrelor infusion, when ticagrelor plasma exposure was measured to bein average 1.4 μM, mice were given an i.v. bolus of 250 mg/kgTICA0212/MEDI2452. At 5, 30, and 60 min post TICA0212/MEDI2452administration mice were sacrificed and blood samples were collected. Inthis study the ADP-induced aggregation response was measured for 6 minand data expressed as the mean area under the curve of aggregation unit(AU) recorded over time (AU*min).

In carrying out the impedance aggregometry assay, mice were sacrificedand blood samples were collected into 7 μM hirudin. Blood (175 μL) wasadded to pre-heated NaCl₂ (37° C., 175 μL) in the Multiplate mini testcells and mixed for 3 min before addition of ADP, 12 μL, to a finalconcentration of 6.5 μM. The disposable Multiplate mini test cellscontain a stir bar and have two separate pair of electrodes that areimmersed into the blood sample.

When agonist (ADP) is added and shear is induced by stirring, plateletswill start to adhere and aggregate onto the electrodes. This results inincreased impedance over the electrodes, which is recorded continuouslyover time by the Multiplate impedance aggregometer (DynaByte, Munchen,Germany)

Ticagrelor treatment induced nearly complete inhibition of ADP-inducedaggregation as the mean (n=4) aggregation response was reduced from 432to 2, from 474 to 6 and from 494 to 14 AU*min at 5, 30, and 60 min afterticagrelor infusion and PBS bolus, respectively (FIG. 9 ).TICA0212/MEDI2452 mediated reversal of in vivo ticagrelor mediatedinhibition as the mean (n=4) aggregation response increased from 2 to147, from 6 to 448 and from 14 to 413 AU*min at 5, 30, and 60 minutesafter ticagrelor infusion and TICA0212 bolus, respectively (FIG. 9 ). Noreversal was present 30 minutes post administration of the isotypecontrol as the mean (n=4) aggregation response remained unchanged from 6to 4 AU*min.

This data shows that TICA0212/MEDI2452 can restore rapidly andeffectively ADP-induced platelet aggregation when given as an i.v. bolusto mice dosed to a ticagrelor plasma concentration of 1.4 μM thatprovided complete inhibition of ADP-induced aggregation.

Example 12: Mouse Bleeding in Ticagrelor-Treated Mice

As one of the intended indications for TICA0212/MEDI2452 is as anantidote for ticagrelor patients requiring urgent surgery,TICA0212/MEDI2452 was evaluated in a mouse bleeding experiment designedto mimic the clinical setting were complete reversal should be achievedbefore initiation of surgery.

Mice were pre-treated with a continuous infusion of ticagrelor, 300μg/kg/min or vehicle for 20 minutes. After stop of infusion, t=0, abolus dose of TICA0212/MEDI2452 (600 mg/kg) or vehicle (histidinesucrose buffer) was given over 45 seconds and at t=30 minutes bleedingwas induced by cutting 5 mm from the tip of the tail. The tip of thetail was rinsed with water (2 mL/min) and the blood and water mixturewas collected in a small chamber, where a stirrer mixed the fluid toenhance haemolysis and establish a homogeneous solution. Lighttransmission, at 525 nm, was recorded for 30 minutes when terminal bloodsamples were collected from the abdominal aorta for plateletaggregation. Light transmittance was transformed to absorbance and usedto calculate blood loss as area under the absorbance curve (AUC,absorbance*s) and total bleeding time (BT, s) by plotting absorbanceovertime. All transmittance below 95% was defined as bleeding. Samplesfor platelet aggregation and total and free plasma exposure were alsocollected at end of ticagrelor infusion (t=0) and at time of tail cut(t=30 minutes).

Studies were approved by the ethical committee for animal research atthe University of Goteborg, Sweden. Mice were anaesthetised withisofluran gas (Forene®, Abbot Scandinavia AB, Sweden). A catheter wasinserted in the left jugular vein for administration of vehicle or drug.The body temperature was maintained at 38° C. by external heating.

To translate the effect of TICA0212/MEDI2452 on platelet aggregation toa potential effect on bleeding, a prophylactic mouse tail bleeding studywas performed. Ticagrelor was infused to a mean total ticagrelor and TAMplasma concentration of 7.6 and 0.3 μM, respectively, to provide asignificant drug-dependent bleeding window. Immediately after stoppingthe ticagrelor infusion TICA0212/MEDI2452 was dosed as a single bolus of600 mg/kg over 30 seconds. After 30 minutes, ADP-induced aggregation wasfully normalized and the mean free plasma concentration of ticagrelorwas reduced from 4.7 nM to below 0.03 nM (lower limit ofquantification). Bleeding was initiated by a tail cut and monitored for30 minutes. In vehicle treated mice the mean total ticagrelor and TAMplasma concentration at time of tail cut were 2.4 and 0.6 μM. The totalblood loss and bleeding time over 30 minutes was significantly (p<0.05)enhanced by ticagrelor by about 3.8-fold and 1.6-fold, respectively.TICA0212/MEDI2452 significantly (p<0.05) reversed blood loss andbleeding time relative ticagrelor alone and back to a level notsignificantly different from mice not treated with ticagrelor (FIG. 12). In this prophylactic setting TICA0212/MED12452 normalized ticagrelordependent bleeding. The onset time of 30 minutes translated from the exvivo model to this in vivo model demonstrating TICA0212/MEDI2452 cannormalise both blood loss and bleeding time to that of mice not treatedwith ticagrelor.

Example 13: Total Plasma Concentration of Ticagrelor and TAM

Blood samples were collected in tubes with EDTA anticoagulant andcentrifuged for 5 min at 10000×g at room temperature to prepare plasma.The plasma concentration of ticagrelor and TAM were determined byprotein precipitation and liquid chromatography with tandem massspectrometry (LC-MS/MS) as described in published method [Sillén H., etal., J Chromatogr B Analyt Technol Biomed Life Sci 2010; 878:2299-306]with the following deviations. Plasma, 50 μL, was protein precipitatedwith 180 μl internal standard (D7-ZD6140) in acetonitrile. The liquidchromatographic system and mass spectrometer was an Acquity UltraPerformance LC coupled to a Xevo TQ-S mass spectrometer from Waters.Chromatographic separation was achieved on an Aquity UPLC® BEH C18column (2.1×50 mm, particle size 1.7 μm). Negative electrosprayionization was utilized. Eluent A was water with 10 mmol/L ammoniumacetate, pH5 and eluent B was acetonitrile with 10 mmol/L ammoniumacetate. Injection volume was 1 to 5 μL and the analytical gradientstarted with 4% B, increasing to 95% in 1.5 min, remaining until 2.3min, before returning to initial conditions within 2.4 min followed by0.3 min re-equilibration. No quality control samples were used in theanalysis. The lower limit of quantification (LLOQ) was 0.005 μmol/L andthe calibration range was 0.005 to 15.0 μM.

The total plasma concentrations of ticagrelor and TAM in presence ofTICA0212/MED12452 were determined by adding 1% formic acid (FA: Sample,1:5) followed by protein precipitation and LC-MS/MS as described above.Formic acid was added to the sample to facilitate dissociation ofticagrelor and TICA0212/MEDI2452.

Example 14: Ticagrelor Free Plasma Concentration

The method was optimized based on a previous published method [SillénH., et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Aug. 1;879(23):2315-22]. Dialysis membranes (Spectrum Laboratories, Inc) whichallows molecules with a mass below 6 to 8 kDa to pass through, wassoaked in ELGA water for 10 to 15 min and placed between the dialysisplate halves (in house made). Plasma, 130 μL, was added to one side ofthe dialysis plate and 130 μL phosphate buffer, pH 7.0, was added to theopposite side. The wells on both sides were sealed with lids andaluminum plates were put on top on each side of the plate to avoidleakage. The plate was then placed vertical on an orbital shaker at 100rpm/min at 37° C. for 24 h. The dialysis was terminated by transferringof 50 μL retentate from plasma side and 75 μL dialysate from buffer sideto a protein LoBind PCR clean 96 deep-well plate containing 150 μLrespective 75 μL internal standard (D7-ZD6140) in acetonitrile. Theplate was mixed for 1 min and then centrifuged for 20 min, 1500×g at 4°C. After centrifugation 50 μL supernatant from the precipitatedretentate was transferred and diluted with 50p ELGA H₂O before LC-MS/MS(Acquity Ultra Performance LC coupled to a Xevo TQ-S mass spectrometer,Waters) analysis. No quality control samples were used in the analysis.Calibration range for retentate was 0.4 to 1000 nmol/L and for dialysate0.003 to 50 nmol/L. The LLOQ for ticagrelor in dialysate was 0.03nmol/L.

Table 9 below provides an overview of the exemplary antibodies describedin the above Examples and used to illustrate certain embodiments of thetechnology disclosed herein.

TABLE 9 Summary of Antibody/scFv/Fab Sequences Antibody Ref SequenceSequence TICA0010 V_(H) DNA GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC(SEQ ID NO: 1) CTGGGGGGTC CCTGAGACTC TCCTGTGCAG CCTCTGGATTCACCTTTAGC AGCTATGCCA TGAGCTGGGT CCGCCAGGCTCCAGGGAAGG GGCTGGAGTG GGTCTCAGCT ATTAGTGGTAGTGGTGGTAG CACATACTAC GCAGACTCCG TGAAGGGCCGGTTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTATCTGCAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCCGTGTATTACTGTGC AACAGAGTAC GACCTGCAAC GGCCTTTCGGGTTTGACTTC TGGGGCAAGG GGACAATGGT CACCGTCTCG AGT TICA0010 V_(H)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI (SEQ ID NO: 2)SGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEYDL QRPFGFDFWGKGTMVTVSSTICA0010 V_(H) CDR1 SYAMS (SEQ ID NO: 3) TICA0010 V_(H) CDR2AISGSGGSTYYADSVKG (SEQ ID NO: 4) TICA0010 V_(H) CDR3 EYDLQRPFGFDF(SEQ ID NO: 5) TICA0010 V_(L) DNATCCTATGTGC TGACTCAGCC ACCCTCAGCG TCTGGGGCCC (SEQ ID NO: 6)CCGGGCAGAG GGCTACCATC TCCTGCTCTG GAAGCAGCTCCAACATCGGA AGTAATCTTG TGAACTGGTA CCAACAATTCCCAGGAGAGG CCCCCAAGCT CCTCATCTTT AGTGACAATCAACGACCCTC AGGGGTCCCT GACCGATTCT CTGGCTCCAGGTCTGGCACC TCAGCCTCCC TGGCCATCAG TGGGCTCCAGTCCGAGGATG AGGCTGATTA TTACTGTGCA ACGTGGGATGACAGACTGGA TGGTTATGTG GTATTCGGCG GAGGGACCAA GCTGACCGTC CTATICA0010 V_(L) SYVLTQFPSASGAPGQRATISCSGSSSNIGSNLVNWYQQFPGEAPKLLIFS(SEQ ID NO: 7) DNQRPSGVPDRFSGSRSGTSASLAISGLQSEDEADYYCATWDDRLDGYVVFGGGTKLTVL TICA0010 V_(L) CDR1 SGSSSNIGSNLVN (SEQ ID NO: 8)TICA0010 V_(L) CDR2 SDNQRPS (SEQ ID NO: 9) TICA0010 V_(L) CDR3ATWDDRLDGYVV (SEQ ID NO: 10) TICA0049 V_(H) DNACAGGTACAGC TGCAGCAGTC AGGGGCTGAG GTGAAGAAGC (SEQ ID NO: 11)CTGGGGCCTC AGTGAAGGTT TCCTGCAAGG CTTCTGGATACACCTTCATT ACCTATGGTA TTCACTGGGT GCGCCAGGCCCCCGGACAAG GGCTTGAGTG GATGGGATGG ATCGACCCCGGGCATGGTTA CACAAAATAT TCACAGAAGT TCCAGGGCAGAGTCACCATT ACCAGGGACA CATCCGCGAG CACAGCCTACATGGAGATGA GCAGCCTCAG ATCTGAAGAC ACGGCTGTGTATTACTGTGC GAGAGCGGAC CTGGGTGACT ACTGGGGCCG GGGAACCCTG GTCACCGTCT CGAGTTICA0049 V_(H) QVQLQQSGAEVKKPGASVKVSCKASGYTFITYGIHWVRQAPGQGLEWMGWI(SEQ ID NO: 12) DPGHGYTKYSQKFQGRVTITRDTSASTAYMEMSSLRSEDTAVYYCARADLGDYWGRGTLVTVSS TICA0049 V_(H) CDR1 TYGIH (SEQ ID NO: 13)TICA0049 V_(H) CDR2 WIDPGHGYTKYSQKFQG (SEQ ID NO: 14)TICA0049 V_(H) CDR3 ADLGDY (SEQ ID NO: 15) TICA0049 V_(L) DNACAGTCTGTCG TGACGCAGCC GCCCTCAGTG TCTGCGGCCC (SEQ ID NO: 16)CAGGACAGAA GGTCACCATC TCCTGCTCTG GAAGCAGCTCCAACATTGGG AAGAATTATG TTTCCTGGTT CCAGCAGCTCCCAGGTACAG CCCCCAAACT CCTCATTTAT GACAATCATAAGCGACCCTC AGGGATTCCT GACCGATTCT CTGCCTCCAAGTCTGGCACG TCAGCCACCC TGGTCATCTC CGGTCTCCAGACTGGGGACG AGGCCCATTA TTACTGCGGA ACATGGGATACCAGACTGAG TGCTGGGGTG TTCGGCGGAG GGACCAAGGT CACCGTCCTA TICA0049 V_(L)QSVVTQPPSVSAAPGQKVTISCSGSSSNIGKNYVSWFQQLPGTAPKLLIYD (SEQ ID NO: 17)NHKRPSGIPDRFSASKSGTSATLVISGLQTGDEAHYYCGTWDTRLSAGVFG GGTKVTVLTICA0049 V_(L) CDR1 SGSSSNIGKNYVS (SEQ ID NO: 18) TICA0049 V_(L) CDR2DNHKRPS (SEQ ID NO: 19) TICA0049 V_(L) CDR3 GTWDTRLSAGV (SEQ ID NO: 20)TICA0053 V_(H) DNA GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC(SEQ ID NO: 21) CTGGGGGGTC CCTGAGACTC TCCTGTGCAG CCTCTGGATTCACCTTTAGC AGCTATGCCA TGAGCTGGGT CCGCCAGGCTCCAGGGAAGG GGCTGGAGTG GGTCTCAGCT ATTAGTGGTAGTGGTGGTAG CACATACTAC GCAGACTCCG TGAAGGGCCGGTTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTATCTGCAAATGA ACAGCCTGAG AGCCGAGGAC ACGGCCGTGTATTACTGTGG CCATGATAGT AGTGGTTACT CCTACTCCTTTGACTTCTGG GGGCGGGGGA CCACGGTCAC CGTCTCGAGT TICA0053 V_(H)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI (SEQ ID NO: 22)SGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGHDSSG YSYSFDFWGRGTTVTVSSTICA0053 V_(H) CDR1 SYAMS (SEQ ID NO: 23) TICA0053 V_(H) CDR2AISGSGGSTYYADSVKG (SEQ ID NO: 24) TICA0053 V_(H) CDR3 DSSGYGYSFDF(SEQ ID NO: 25) TICA0053 V_(L) DNACAGTCTGTGT TGACGCAGCC GCCCTCAGCG TCTGGGACCC (SEQ ID NO: 26)CCGGGCAGAG GGTCACCATC TCTTGTTCTG GCAACATCTCCAACATCGGA AGTAACACTG TCAACTGGTA TCAACACGTCCCAGGAGCGG CCCCCAGACT CCTCATCTAT GTTAATGATCAGCGGCCGTC AGGGGTCCCT GACCGATTCT CTGGCTCCAAGTCTGGCACC TCAGCCTCCC TGGCCATCAG TGGGCTCCAGTCTGAAGATG AGGCTGATTA TTACTGTGCA ACGTGGGATGACACCCTGAA TGGAGGGGTC TTCGGCGGAG GGACCAAGCT GACCGTCCTA TICA0053 V_(L)QSVLTQPPSASGTPGQRVTISCSGNISNIGSNTVNWYQHVPGAAPRLLIYV (SEQ ID NO: 27)NDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDTLNGGVFG GGTKLTVLTICA0053 V_(L) CDR1 SGNISNIGSNTVN (SEQ ID NO: 28) TICA0053 V_(L) CDR2VNDQRPS (SEQ ID NO: 29) TICA0053 V_(L) CDR3 ATWDDTLNGGV (SEQ ID NO: 30)TICA0072 V_(H) DNA CAGGTGCAGC TGCAGGAGTC CGGGGCTGAG GTGAAGAAGC(SEQ ID NO: 31) CTGGGTCCTC GGTGAGGGTC TCCTGCAAGG CTTCTGGAGGCACCTTCGAC AGTTATAGTA TCCATTGGGT GCGCCAGGCCCCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTGCCTTTGGGAC ATTAAGCAGC GCACAGGACT TCCAGGCCAGAGTCACCATT AGCGCGGACA AGTCCACGAG CACAGCCTATATGGAGCTGA GCGGCCTGAG ATCTGAGGAC ACGGCCGTATATTACTGTGC GAGAGGGTCC CATCTTTACG ATTTTTGGAGTGCTTCTCAT CCCCCCAATG ATGCTCTTGC TATTTGGGGCCAAGGAACCC TGGTCACCGT CTCGAGT TICA0072 V_(H)QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIHWVRQAPGQGLEWMGGI (SEQ ID NO: 32)IPAFGTLSSAQDFQARVTISADKSTSTAYMELSGLRSEDTAVYYCARGSHLYDFWSASHPPNDALAIWGQGTLVTVSS TICA0072 V_(H) CDR1 SYSIH (SEQ ID NO: 33)TICA0072 V_(H) CDR2 GIIPAFGTLSSAQDFQA (SEQ ID NO: 34)TICA0072 V_(H) CDR3 GSHLYDFWSASHPPNDALAI (SEQ ID NO: 35)TICA0072 V_(L) DNA CAGTCTGTCG TGACGCAGCC GCCCTCAGTG TCTGCGGCCC(SEQ ID NO: 36) CAGGACAGAA GGTCACCATC TCCTGCTCTG GAAGCAACTCCGACATTGGC AACAATTATG TGTCGTGGTA CCAACAGCTCCCAGGAACAG CCCCCAAACT CCTCATTTAT GACAATAATAAACGACCCTC AGGGATTCCT GACCGATTCT CTGGCTCCAAGTCTGGCACG TCAGCCACCC TGGCCATCAC CGGACTCCAGGCTGGGGACG AGGCCGATTA TTACTGCGGG ACATGGGATATCAGCCTGAG CGCTGGCTTG TTCGGCGGAG GGACCAAGGT CACCGTCCTA TICA0072 V_(L)QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVSWYQQLPGTAPKLLIYD (SEQ ID NO: 37)NNKRPSGIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWDISLSAGLFG GGTKVTVLTICA0072 V_(L) CDR1 SGSNSDIGNNYVS (SEQ ID NO: 38) TICA0072 V_(L) CDR2DNNKRPS (SEQ ID NO: 39) TICA0072 V_(L) CDR3 GTWDISLSAGL (SEQ ID NO: 40)TICA0159 V_(H) DNA CAGGTGCAGC TGCAGGAGTC CGGGGCTGAG GTGAAGAAGC(SEQ ID NO: 41) CTGGGTCCTC GGTGAGGGTC TCCTGCAAGG CTTCTGGAGGCACCTTCGAC AGTTATAGTA TCCATTGGGT GCGCCAGGCCCCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTGCCTTTGGGAC ATTAAGCAGC GCACAGGACT TCCAGGCCAGAGTCACCATT AGCGCGGACA AGTCCACGAG CACAGCCTATATGGAGCTGA GCGGCCTGAG ATCTGAGGAC ACGGCCGTATATTACTGTGC GAGAGGGAGC TTCGACTACA GGTTTTGGAGTGCTTCTCAT CCCCCCAATG ATGCTCTTGC TATTTGGGGCCAAGGAACCC TGGTCACCGT CTCGAGT TICA0159V_(H)QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIHWVRQAPGQGLEWMGGI (SEQ ID NO: 42)IPAFGTLSSAQDFQARVTISADKSTSTAYMELSGLRSEDTAVYYCARGSFDYRFWSASHPPNDALAIWGQGTLVTVSS TICA0159 V_(H) CDR1 SYSIH (SEQ ID NO: 43)TICA0159 V_(H) CDR2 GIIPAFGTLSSAQDFQA (SEQ ID NO: 44)TICA0159 V_(H) CDR3 GSFDYRFWSASHPPNDALAI (SEQ ID NO: 45)TICA0159 V_(L) DNA CAGTCTGTCG TGACGCAGCC GCCCTCAGTG TCTGCGGCCC(SEQ ID NO: 46) CAGGACAGAA GGTCACCATC TCCTGCTCTG GAAGCAACTCCGACATTGGC AACAATTATG TGTCGTGGTA CCAACAGCTCCCAGGAACAG CCCCCAAACT CCTCATTTAT GACAATAATAAACGACCCTC AGGGATTCCT GACCGATTCT CTGGCTCCAAGTCTGGCACG TCAGCCACCC TGGCCATCAC CGGACTCCAGGCTGGGGACG AGGCCGATTA TTACTGCGGG ACATGGGATATCAGCCTGAG CGCTGGCTTG TTCGGCGGAG GGACCAAGGT CACCGTCCTA TICA0159 V_(L)QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVSWYQQLPGTAPKLLIY D (SEQ ID NO: 47)NNKRPSGIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWDISLSAGLFG GGTKVTVLTICA0159 V_(L) CDR1 SGSNSDIGNNYVS (SEQ ID NO: 48) TICA0159 V_(L) CDR2DNNKRPS (SEQ ID NO: 49) TICA0159 V_(L) CDR3 GTWDISLSAGL (SEQ ID NO: 50)TICA0162 V_(H) DNA CAGGTGCAGC TGCAGGAGTC CGGGGCTGAG GTGAAGAAGC(SEQ ID NO: 51) CTGGGTCCTC GGTGAGGGTC TCCTGCAAGG CTTCTGGAGGCACCTTCGAC AGTTATAGTA TCCATTGGGT GCGCCAGGCCCCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTGCCTTTGGGAC ATTAAGCAGC GCACAGGACT TCCAGGCCAGAGTCACCATT AGCGCGGACA AGTCCACGAG CACAGCCTATATGGAGCTGA GCGGCCTGAG ATCTGAGGAC ACGGCCGTATATTACTGTGC GAGAGGCTCC TTCGACTACT ACTTTTGGAGTGCTTCTCAT CCCCCCAATG ATGCTCTTGC TATTTGGGGCCAAGGAACCC TGGTCACCGT CTCGAGT TICA0162 V_(H)QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIHWVRQAPGQGLEWMGGI (SEQ ID NO: 52)IPAFGTLSSAQDFQARVTISADKSTSTAYMELSGLRSEDTAVYYCARGSFDYYFWSASHPPNDALAIWGQGTLVTVSS TICA0162 V_(H) CDR1 SYSIH (SEQ ID NO: 53)TICA0162 V_(H) CDR2 GIIPAFGTLSSAQDFQA (SEQ ID NO: 54)TICA0162 V_(H) CDR3 GSFDYYFWSASHPPNDALAI (SEQ ID NO: 55)TICA0162 V_(L) DNA CAGTCTGTCG TGACGCAGCC GCCCTCAGTG TCTGCGGCCC(SEQ ID NO: 56) CAGGACAGAA GGTCACCATC TCCTGCTCTG GAAGCAACTCCGACATTGGC AACAATTATG TGTCGTGGTA CCAACAGCTCCCAGGAACAG CCCCCAAACT CCTCATTTAT GACAATAATAAACGACCCTC AGGGATTCCT GACCGATTCT CTGGCTCCAAGTCTGGCACG TCAGCCACCC TGGCCATCAC CGGACTCCAGGCTGGGGACG AGGCCGATTA TTACTGCGGG ACATGGGATATCAGCCTGAG CGCTGGCTTG TTCGGCGGAG GGACCAAGGT CACCGTCCTA TICA0162 V_(L)QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVSWYQQLPGTAPKLLIYD (SEQ ID NO: 57)NNKRPSGIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWDISLSAGLFG GGTKVTVLTICA0162 V_(L) CDR1 SGSNSDIGNNYVS (SEQ ID NO: 58) TICA0162 V_(L) CDR2DNNKRPS (SEQ ID NO: 59) TICA0162 V_(L) CDR3 GTWDISLSAGL (SEQ ID NO: 60)TICA0152 V_(H) DNA CAGGTGCAGC TGCAGGAGTC CGGGGCTGAG GTGAAGAAGC(SEQ ID NO: 61) CTGGGTCCTC GGTGAGGGTC TCCTGCAAGG CTTCTGGAGGCACCTTCGAC AGTTATAGTA TCCATTGGGT GCGCCAGGCCCCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTGCCTTTGGGAC ATTAAGCAGC GCACAGGACT TCCAGGCCAGAGTCACCATT AGCGCGGACA AGTCCACGAG CACAGCCTATATGGAGCTGA GCGGCCTGAG ATCTGAGGAC ACGGCCGTATATTACTGTGC GAGAGGGTCC CATCTTTACG ATTTTTGGAGTGCTTCTCAT CCCCCCAATG ATGCTCTTGC TATTTGGGGCCAAGGAACCC TGGTCACCGT CTCGAGT TICA0152 V_(H)QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIHWVRQAPGQGLEWMGGI (SEQ ID NO: 62)IPAFGTLSSAQDFQARVTISADKSTSTAYMELSGLRSEDTAVYYCARGSHLYDFWSASHPPNDALAIWGQGTLVTVSS TICA0152 V_(H) CDR1 SYSIH (SEQ ID NO: 63)TICA0152 V_(H) CDR2 GIIPAFGTLSSAQDFQA (SEQ ID NO: 64)TICA0152 V_(H) CDR3 GSHLYDFWSASHPPNDALAI (SEQ ID NO: 65)TICA0152 V_(L) DNA CAGTCTGTCG TGACGCAGCC GCCCTCAGTG TCTGCGGCCC(SEQ ID NO: 66) CAGGACAGAA GGTCACCATC TCCTGCTCTG GAAGCAACTCCGACATTGGC AACAATTATG TGTCGTGGTA CCAACAGCTCCCAGGAACAG CCCCCAAACT CCTCATTTAT GACAATAATAAACGACCCTC AGGGATTCCT GACCGATTCT CTGGCTCCAAGTCTGGCACG TCAGCCACCC TGGCCATCAC CGGACTCCAGGCTGGGGACG AGGCCGATTA TTACTGCGGG ACATGGCTGTACGACCGGGC CGTCGGCTTG TTCGGCGGAG GGACCAAGGT CACCGTCCTA TICA0152 V_(L)QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVSWYQQLPGTAPKLLIYD (SEQ ID NO: 67)NNKRPSGIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWLYDRAVGLFG GGTKVTVLTICA0152 V_(L) CDR1 SGSNSDIGNNYVS (SEQ ID NO: 68) TICA0152 V_(L) CDR2DNNKRPS (SEQ ID NO: 69) TICA0152 V_(L) CDR3 GTWLYDRAVGL (SEQ ID NO: 70)TICA0212/MEDI2452 CAGGTGCAGC TGCAGGAGTC CGGGGCTGAG GTGAAGAAGC V_(H) DNACTGGGTCCTC GGTGAGGGTC TCCTGCAAGG CTTCTGGAGG (SEQ ID NO: 71)CACCTTCGAC AGTTATAGTA TCCATTGGGT GCGCCAGGCCCCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTGCCTTTGGGAC ATTAAGCAGC GCACAGGACT TCCAGGCCAGAGTCACCATT AGCGCGGACA AGTCCACGAG CACAGCCTATATGGAGCTGA GCGGCCTGAG ATCTGAGGAC ACGGCCGTATATTACTGTGC GAGAGGCTCC TTCGACTACT ACTTTTGGAGTGCTTCTCAT CCCCCCAATG ATGCTCTTGC TATTTGGGGCCAAGGAACCC TGGTCACCGT CTCGAGT TICA0212/MEDI2452QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIHWVRQAPGQGLEWMGGI V_(H)IPAFGTLSSAQDFQARVTISADKSTSTAYMELSGLRSEDTAVYYCARGSFD (SEQ ID NO: 72)YYFWSASHPPNDALAIWGQGTLVTVSS TICA0212/MEDI2452 SYSIH V_(H) CDR1(SEQ ID NO: 73) TICA0212/MEDI2452 GIIPAFGTLSSAQDFQA V_(H) CDR2(SEQ ID NO: 74) TICA0212/MEDI2452 GSFDYYFWSASHPPNDALAI V_(H) CDR3(SEQ ID NO: 75) TICA0212/MEDI2452CAGTCTGTCG TGACGCAGCC GCCCTCAGTG TCTGCGGCCC V_(L) DNACAGGACAGAA GGTCACCATC TCCTGCTCTG GAAGCAACTC (SEQ ID NO: 76)CGACATTGGC AACAATTATG TGTCGTGGTA CCAACAGCTCCCAGGAACAG CCCCCAAACT CCTCATTTAT GACAATAATAAACGACCCTC AGGGATTCCT GACCGATTCT CTGGCTCCAAGTCTGGCACG TCAGCCACCC TGGCCATCAC CGGACTCCAGGCTGGGGACG AGGCCGATTA TTACTGCGGG ACATGGCTGTACGACCGGGC CGTCGGCTTG TTCGGCGGAG GGACCAAGGT CACCGTCCTA TICA0212/MEDI2452QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVSWYQQLPGTAPKLLIYD V_(L)NNKRPSGIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWLYDRAVGLFG (SEQ ID NO: 77)GGTKVTVL TICA0212/MEDI2452 SGSNSDIGNNYVS V_(L) CDR1 (SEQ ID NO: 78)TICA0212/MEDI2452 DNNKRPS V_(L) CDR2 (SEQ ID NO: 79) TICA0212/MEDI2452GTWLYDRAVGL V_(L) CDR3 (SEQ ID NO: 80)

REFERENCES AND INCORPORATION BY REFERENCE

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All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific aspects of the subject disclosure have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the disclosure will become apparent to those skilled inthe art upon review of this specification and the claims below. The fullscope of the disclosure should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

We claim:
 1. An antibody or a fragment thereof that specifically binds acyclopentyltriazolopyrimidine compound of the Formula (Ia):

wherein R₁ is selected from the group consisting of C₁-C₆ alkoxy andC₁-C₆ alkylthio; R₂ is selected from the group consisting of H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl, and substituted C₃-C₆cycloalkyl; and R₃ is selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆ alkoxy, and C₁-C₆ alkanol; wherein the antibody or afragment thereof comprises the complementarity-determining regions (CDR)of: SEQ ID NO:73 (VH CDR1), SEQ ID NO:74 (VH CDR2), SEQ ID NO:75 (VHCDR3), SEQ ID NO:78 (VL CDR1), SEQ ID NO:79 (VL CDR2), and SEQ ID NO:80(VL CDR3).
 2. The antibody or fragment thereof of claim 1, wherein theantibody or fragment thereof is selected from a monoclonal antibody, ahumanized antibody, a human antibody, a single chain Fv (scFv), a Fab, aF(ab′)₂, a single chain diabody, and an antibody mimetic.
 3. Theantibody or fragment thereof of claim 1, wherein the antibody orfragment thereof binds ticagrelor or an active metabolite thereof. 4.The antibody or fragment thereof of claim 3, wherein the antibody orfragment thereof neutralizes the antiplatelet effect of ticagrelor or anactive metabolite thereof.
 5. The antibody or fragment thereof of claim4, wherein the antibody or fragment thereof neutralizes the antiplateleteffect of ticagrelor or an active metabolite thereof within about 60minutes after administration to a patient.
 6. The antibody or fragmentthereof of claim 5, wherein the antibody or fragment thereof neutralizesthe antiplatelet effect of ticagrelor or an active metabolite thereofwithin about 30 minutes after administration to a patient.
 7. Theantibody or fragment thereof of claim 3, wherein the antibody orfragment thereof restores ADP-induced platelet aggregation in thepresence of ticagrelor or an active metabolite of ticagrelor.
 8. Theantibody or fragment thereof of claim 3, wherein the antibody orfragment thereof inhibits the effect of ticagrelor or an activemetabolite thereof on the P2Y12 receptor.
 9. The antibody or fragmentthereof of claim 3, wherein the antibody or fragment thereof bindsticagrelor or an active metabolite thereof with an equilibrium KD ofabout 50 nM or lower.
 10. The antibody or fragment thereof of claim 1,wherein the antibody or fragment thereof has an in vivo half-life ofabout 4-24 hours.
 11. An antibody or fragment thereof comprising thecomplementarity-determining regions (CDR) of: SEQ ID NO:73 (VH CDR1),SEQ ID NO:74 (VH CDR2), SEQ ID NO:75 (VH CDR3), SEQ ID NO:78 (VL CDR1),SEQ ID NO:79 (VL CDR2), and SEQ ID NO:80 (VL CDR3).
 12. The antibody orfragment thereof of claim 11, wherein the antibody or fragment thereofis selected from a monoclonal antibody, a humanized antibody, a humanantibody, a single chain Fv (scFv), a Fab, a F(ab′)2, a single chaindiabody, and an antibody mimetic.
 13. The antibody or fragment thereofof claim 11, wherein the antibody or fragment thereof binds ticagreloror an active metabolite thereof.
 14. A Fab comprising thecomplementarity-determining regions (CDR) of: SEQ ID NO:73 (VH CDR1),SEQ ID NO:74 (VH CDR2), SEQ ID NO:75 (VH CDR3), SEQ ID NO:78 (VL CDR1),SEQ ID NO:79 (VL CDR2), and SEQ ID NO:80 (VL CDR3).
 15. The Fab of claim14, wherein the antibody or fragment thereof binds ticagrelor or anactive metabolite thereof.